Noncoding Ribonucleic Acids (RNAs) May Improve Response to Immunotherapy in Pancreatic Cancer

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is the seventh most common cause of cancer-related mortality. Despite different methods of treatment, nearly more than 90% of patients with PDAC die shortly after diagnosis. Contrary to promising results in other cancers, immune checkpoint inhibitors (ICIs) showed limited success in PDAC. Recent studies have shown that noncoding RNAs (ncRNAs) are extensively involved in PDAC cell–immune cell interaction and mediate immune evasion in this vicious cancer. PDAC cells recruit numerous ncRNAs to widely affect the phenotype and function of immune cells through various mechanisms. For instance, PDAC cells upregulate miR-301a and downregulate miR-340 to induce M2 polarization of macrophages or overexpress miR-203, miR-146a, and miR-212-3p to downregulate toll-like receptor 4 (TLR4), CD80, CD86, CD1a, major histocompatibility complex (MHC) II, and CD83, thereby evading recognition by dendritic cells. By downregulating miR-4299 and miR-153, PDAC cells can decrease the expression of NK group 2D (NKG2D) and MHC class I chain-related molecules A and B (MICA/B) to blunt the natural killer (NK) cell response. PDAC cells also highly express lncRNA AL137789.1, hsa_circ_0046523, lncRNA LINC00460, and miR-155-5p to upregulate immune checkpoint proteins and escape T cell cytotoxicity. On the other hand, ncRNAs derived from suppressive immune cells promote proliferation, invasion, and drug resistance in PDAC cells. ncRNAs can be applied to overcome resistance to ICIs, monitor the immune microenvironment of PDAC, and predict response to ICIs. This Review article comprehensively discusses recent findings regarding the roles of ncRNAs in the immune evasion of PDAC.

Contrary to its low prevalence, PDAC is the seventh most common cause of cancer-related mortality, reminding its high mortality rate and poor prognosis.¹ Despite different methods of treatment, more than 90% of patients with PDAC die shortly after diagnosis.¹ Between 1990 and 2017, the age-standardized incidence rate of PDAC increased from 5.0 per 100,000 person-years to 5.7 per 100,000 person-years, the annual death from PDAC increased by 2.3 times, and disability-adjusted life year (DALYs) due to PDAC increased by 2.1 times.² The 5-year survival rate of PDAC is approximately 10% in the USA, and patients often present with advanced cancer due to the lack of characteristic symptoms in the early stages of the disease.³

Currently, surgical resection is the only chance for cure in the early stages of the disease, while chemotherapy regimens can partly prolong survival in later stages.^3,4 Particularly, FOLFIRINOX consisting of leucovorin (folinic acid), fluorouracil, irinotecan, and oxaliplatin, and the combination of gemcitabine and nab-paclitaxel can improve the prognosis of patients with advanced PDAC.³⁻⁶

Contrary to all efforts, the survival rate of patients remained unsatisfactory, and there is great space for improvement. Therefore, clinical trials are now measuring the combination of chemotherapy and immunotherapy for advanced PDAC.⁷ Recent clinical trials mostly focused on ICIs, such as pembrolizumab, nivolumab, and ipilimumab, and cancer vaccines for immunotherapy of PDAC.⁷⁻¹⁰ The addition of immunotherapy to chemotherapy partly improved patients’ 1-year overall survival;⁹ however, it could not make a breakthrough in the treatment of PDAC and barely increased patients’ survival for 2–3 months.⁷⁻⁹

A major proportion of the human genome is transcribed into ncRNAs.¹¹ ncRNAs, particularly microRNAs (miRs), long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs), can modulate gene expression patterns mainly through epigenetic or post-transcriptional modifications.¹²⁻¹⁸ ncRNAs are involved in immune response, regulate different aspects of cancer biology, and can be used to improve the efficacy of immunotherapy in PDAC.¹⁹⁻²² By releasing ncRNAs, cancer cells can alter the function of the immune system and interact with different types of immune cells.^21,22

Here, this review article discusses the role of ncRNAs in the regulation of the immune microenvironment of PDAC and depicts a new method of immunotherapy, which may be more efficacious when used in combination with ICIs. Specifically, the molecular mechanisms linking ncRNAs to the anticancer response of the immune system will be discussed. Moreover, this narrative review introduces potential ncRNAs inducing resistance to immunotherapy in PDAC or helping to predict the response to immunotherapy in PDAC.

Immune Evasion in PDAC

As part of a self-defense mechanism, cancer cells induce a series of adaptive alterations in their microenvironment and dampen the immune response to guarantee their survival and unlimited growth and proliferation.^23,24 Immune cells, particularly T cells, NK cells, and macrophages, can directly remove cancer cells with unfamiliar antigens. Therefore, it is immensely important for cancer cells to reshape their immune microenvironment and evade immune response. The following section explains some of the main immune evasion mechanisms by which PDAC cells escape the immune response. A deeper insight into such mechanisms can help better understand the nature of PDAC and illuminate the need for developing novel strategies for immunotherapy in PDAC.

Specifically, extracellular vesicles are major means of intercellular communication. By releasing extracellular vesicles with numerous mediators and signaling molecules, including ncRNAs, PDAC cells can induce the suppressive phenotype of immune cells or impair the cytotoxic and phagocytic function of mature immune cells.²⁵ Therefore, reprogramming of NK cells can help overcome immune evasion of cancer cells.²⁶

Recently, it has been demonstrated that cancer cells heavily rely on ncRNAs to reshape the anticancer response of the immune system.²⁷ Exosomal ncRNAs can reprogram gene expression patterns in cancer cells, immune cells, and other cells in the tumor microenvironment and promote differentiation into suppressive phenotypes.²⁷ For instance, it has been shown that PDAC cells overexpress and release certain ncRNAs, such as lncRNA AL137789.1, which can suppress T cell cytotoxicity against these cancer cells.²⁷

In addition to PDAC cells, cancer-associated fibroblasts (CAFs) considerably dampen the cytotoxic function of immune cells to provide a safe microenvironment for PDAC proliferation.²⁸ Interestingly, CAFs isolated from PDAC tissues overexpressed programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2) compared with primary skin fibroblasts from healthy donors.²⁹ Besides, CAFs isolated from PDAC tissue upregulated the expression of other immune checkpoint proteins such as T cell immunoglobulin and mucin domain-containing protein 3 (TIM3), programmed death 1 (PD-1), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and lymphocyte activation gene 3 (LAG-3) in cocultured proliferating T-cells and undermined their ability to produce inflammatory cytokines such as interferon gamma (IFN-γ) and tumor necrosis factor α (TNF-α).²⁹ Moreover, CAFs promoted the expression of forkhead box P3 (FOXP3), a T Reg marker, in cocultured unstimulated CD4+ cells.²⁹ It has been observed that similar to PDAC tissue, cultured hypoxic CAFs exhibit an altered expression pattern of ncRNA.³⁰ Specifically, researchers found that hypoxic CAFs have increased expression of miR-21 and miR-210.³⁰ Previous studies illuminated that CAFs can overexpress specific ncRNAs, such as miR-92 and circEIF3K, which can finally upregulate immune checkpoint proteins, such as PD-L1, in cancer cells and enhance tumor immune evasion.^31,32 Luciferase assay indicated that miR-92 can target and downregulate the expression of large tumor suppressor kinase 2 (LATS2), thereby attenuating the interaction between yes-associated protein 1 (YAP1) and LATS2 and weakening the inhibitory effect of LATS2 on YAP1-mediated expression of PD-L1.³¹ Similarly, exosomal circEIF3K released by cancer cells was shown to downregulate miR-214 in cancer cells and attenuate the inhibitory effect of miR-214 on PD-L1 expression.³² In addition, miR-320a released by CAFs was shown to upregulate anti-inflammatory cytokines, such as IL10, in PDAC.³³ miR-320a was shown to target the 3′UTR of phosphatase and tensin homologue (PTEN), thereby promoting the M2 polarization of macrophages and inducing an anti-inflammatory microenvironment.³³ These findings are only instances of how ncRNAs may be involved in the immunosuppressive properties of CAFs.

PDAC cells were shown to release anti-inflammatory chemokines such as C-X-C motif chemokine (CXCL) 5, CXCL9, and CXCL10 to prevent CD8+ T cell proliferation and function in the tumor microenvironment.³⁴ Mechanistically, it was found that CXCL9 can bind to its receptor, C-X-C motif chemokine receptor 3 (CXCR3) on immune cells.³⁵ After binding to CXCL9, CXCR3 activates signal transducer and activator of transcription (STAT)-3, a transcription factor whose activation suppresses CD8+ T cell response and facilitates immune evasion.³⁴ Similarly, it has been shown that patients with PDAC possess higher serum levels of specific adipokines, such as retinol-binding protein-4 (RBP-4) and neutrophil gelatinase-associated lipocalin (NGAL), which may dampen the immune response.^36,37

Previous studies have shown that low CD8+ T cell infiltration and high expression of PD-L1 in PDAC are associated with higher abundances of cancer stem cells, shorter survival, and higher risk of recurrence.³⁸ Similarly, it has been reported that high abundances of both CD8+ T cells and CD4+ T cells can independently predict longer overall survival of patients with PDAC.³⁹ Likewise, Tahkola et al. indicated that a higher abundance of CD3+ and CD8+ immune cells in the PDAC microenvironment is independently associated with prolonged disease-specific survival and overall survival of patients.⁴⁰ In addition, Liu et al. observed that a lower abundance of intratumoral T Reg cells is associated with longer disease-free survival in PDAC.⁴¹ CTLA-4-overexpressing T Reg cells accumulate in the tumor microenvironment and sentinel lymph nodes during PDAC development and progression, respectively.⁴² As an immune checkpoint protein, CTLA-4 on T Reg cells interacts with CD80 on antigen-presenting cells (APCs) and attenuates the cytotoxic function of effector T cells.⁴² On the other hand, PDAC cells can downregulate MHC-I through autophagic degradation, thereby preventing their recognition by immune cells and evading T cell cytotoxicity.⁴³ Interestingly, it has been observed that downregulation of some ncRNAs, such as miR-137, miR-29c, and miR-23b, enhances autophagy in PDAC cells and concomitantly induces resistance to treatment.⁴⁴⁻⁴⁶ Furthermore, forced upregulation of these ncRNAs can inhibit autophagy in PDAC cells and overcome treatment resistance.⁴⁴⁻⁴⁶

Interestingly, it has been unfolded that mutation of Kirsten rat sarcoma virus (KRAS), a major proto-oncogene in PDAC, is a major driver of immune escape in this cancer.⁴⁷ Herein, KRAS-deficient PDAC cells failed to establish a tumor in wild-type mice while forming pancreatic tumor in immune-deficient mice.⁴⁷ KRAS knockout tumor had significantly higher abundances of CD4+, CD8+ T cells, and M1 macrophages in the tumor microenvironment.⁴⁷ In addition, KRAS-deficient PDAC cells had higher expression of chemokines.⁴⁷ Further studies unveiled that KRAS acts through myeloid cell-specific nuclear antigen (MYC) and B-Raf proto-oncogene (BRAF), two downstream oncogenes, to exert its immune-suppressive effects.⁴⁷ Previous studies unraveled that many miRs, such as the let-7 family, miR-18a, miR-143, and miR-31, can target the 3′-UTR of KRAS mRNA and regulate its expression in cancer cells, thereby modulating cancer cell growth, proliferation, invasion, and immune evasion.⁴⁸ Compared with low miR-143 expression, high miR-143 expression was shown to be associated with an increased risk of lymph node involvement and shorter overall survival and disease-free survival in patients with PDAC.⁴⁹

PDAC cells also enhance the M2 polarization of macrophages, which can promote tumor growth and metastasis.⁵⁰ Similarly, it has been observed that CAFs isolated from human PDAC tissues shift the polarization of cocultured macrophages toward the M2 phenotype, which can indirectly promote tumor growth.⁵¹ Using a mice model of PDAC, it has been shown that 85% of macrophages in the tumor microenvironment are M2 macrophages.⁵² Interestingly, PDAC culture supernatant promoted M2 polarization and hindered M1 polarization of naïve macrophages.⁵² Furthermore, the culture supernatant of M2 macrophages promoted in vitro growth of PDAC by 63%.⁵² These findings indicate that PDAC cells and CAFs can escape M1 phenotype-mediated phagocytosis by promoting M2 polarization and benefit from M2 phenotype-mediated growth.⁵² Remarkably, ncRNAs released by PDAC cells and CAFs were found to play a crucial role in shifting macrophage polarization.³³ For instance, CAF-derived exosomal miRNA-320a can promote the M2 polarization of cocultured macrophages.³³ Likewise, exosomal miR-301a released by hypoxic PDAC cells was found to shift macrophage polarization toward the M2 phenotype.⁵³

Similar to macrophages and T cells, NK cells from patients with PDAC show impaired anticancer function.⁵⁴ It has been shown that NK cells from the peripheral blood of PDAC patients have decreased cytotoxic activity, marked downregulation of NKG2D, and low expression levels of IFN-γ but high expression levels of IL-10, which suggests a suppressive phenotype that cannot effectively remove cancer cells.⁵⁴ Likewise, NK cells isolated from PDAC tissues or cocultured with PDAC cells had lower expression of NK cell markers such as CD16 and CD57 and activation markers such as DNAX activation motif-1 (DNAM-1) and NK cell-activating receptor (NKP30).⁵⁴ Interestingly, extracellular vesicles from PDAC inhibited the cytotoxic function of NK cells and markedly downregulated NKG2D, TNF-α, and INF-γ expression by NK cells.⁵⁵ Mechanistically, extracellular vesicles from PDAC contained a high amount of transforming growth factor beta (TGF-β), which activated Smad2/3 in NK cells and weakened NK cell-mediated cytotoxicity.⁵⁵ Activation and nuclear translocation of STAT-3 and Smad2/3 in cancer⁵⁶ can promote the expression of immune checkpoint proteins and suppress immune function.^57,58

Similarly, TP53 mutations play a significant role in the development, progression, and immune evasion of PDAC.⁵⁹ Herein, it was found that TP53 mutations can alter the expression of immune receptors and ligands, such as PD-L1, FAS, and TLRs. In addition, mutated TP53 can interact with many ncRNAs, such as miR-125a, miR-143, and miR-145, to regulate the progression of PDAC.^59,60

The following section, we discuss the role of ncRNAs in different aspects of immune evasion in PDAC and provide a theoretical basis for improving immunotherapy in this cancer.

ncRNAs Can Predict the Immune Microenvironment of PDAC

Identification of novel biomarkers that predict response to immunotherapy can substantially improve therapeutic response, modify the treatment plan, contribute to individualized treatment, screen nonresponders, and prevent the adverse effects of immunotherapy.⁶¹ Herein, Wang et al. developed a prognostic model for PDAC based on 14 tumor mutation burden-related lncRNAs.⁶² Using data from 266 patients with PDAC, they found that patients with high risk scores based on this prognostic model had worse prognoses, low immune scores, and less infiltration of immune cells.⁶² In particular, higher abundances of CD8+ T cells, NK cells, and macrophages were found in the tissue samples of the low-risk group compared with the high-risk group.⁶² Shen et al. developed a prognostic model based on 7 immune-related miRs, namely hsa-miR-550a-3–5p, hsa-miR-3613–5p, hsa-miR-221–3p, hsa-miR-424–5p, hsa-miR-491–3p, hsa-miR-1179, and hsa-miR-3614–3p, which effectively prognosticated overall survival in PDAC.⁶³ They indicated that these miRs mostly controlled dendritic cell infiltration and regulated the expression of immune checkpoint genes such as TNF superfamily member 9 (TNFSF9), TNF receptor superfamily member 9 (TNFRSF9), killer cell immunoglobulin-like receptor 3DL1 (KIR3DL1), hepatitis A virus cellular receptor 2 (HAVCR2), CD80, and CD276.⁶³ Comparing gemcitabine-resistant PDAC cells and their parental cells, Gu et al. identified four differential expressed miRs, including hsa-miR-3178, hsa-miR-485–3p, hsa-miR-574–5p, and hsa-miR-584–5p, which were linked to CD4+ memory T cells and predicted patients’ survival based on the TCGA PAAD data set.⁶⁴ Using normal and PDAC tissues, Felix et al. indicated that the miR-216 family, including miR-216a-3p, miR-216a-5p, miR-216b-3p, and miR-216b-5p, are downregulated in PDAC, whose function was found to be mostly related to innate and adaptive immune response.⁶⁵ Specifically, the miR-216 family was shown to modulate the expression of many CD markers, such as CD22, CD36, CD79A, and CD226, and other surface receptors related to the innate and adaptive immune response.⁶⁵

Ye et al. constructed a prediction model based on seven CAFs-related lncRNAs, including lncRNA CASC8, lncRNA AP005233.2, lncRNA AC090114.2, lncRNA DCST1-AS1, lncRNA AC092171.5, lncRNA AC002401.4, lncRNA AC025048.4, and lncRNA CASC8. The model successfully predicted the overall survival and progression-free survival of PDAC patients.⁶⁶ They indicated that the low-risk group had a significantly higher abundance of CD8+ T cells, dendritic cells, neutrophils, T helper cells, and tumor-infiltrating lymphocytes compared with the high-risk group.⁶⁶ Furthermore, the immune score was higher in the low-risk group than in the high-risk group.⁶⁶ Interestingly, the study indicated that compared with the high-risk group, the low-risk group highly expressed CTLA-4 and PD-1, which makes them ideal candidates for immunotherapy with ICIs.⁶⁶ Xu et al. also constructed a prediction model based on m6A-related lncRNAs to predict patients′ survival in PDAC.⁶⁷ Interestingly, they indicated that the risk score in their study was inversely correlated with genes linked to immune checkpoint blockade such as PD-1, CTLA-4, indoleamine 2,3-dioxygenase (IDO)1, and IDO2.⁶⁷

These findings indicate that ncRNAs not only inform us about the immune microenvironment and prognosis of PDAC but also predict the response to immunotherapy in this cancer.

The Involvement of ncRNAs in Immune Cell Function in PDAC

ncRNAs Modulate Cancer Cell Recognition and Antigen Presentation by Dendritic Cells in PDAC

As a main group of APCs, dendritic cells are deeply involved in cancer cell recognition and initiation and modification of innate and adaptive immune responses.⁶⁸ By identifying neoantigens of PDAC and introducing them to T cells, dendritic cells provoke a vigorous response of adaptive immunity.⁶⁹ Since the competent response of dendritic cells plays a pivotal role in the subsequent removal of cancer cells, many studies have investigated the efficacy of dendritic cell-based immunotherapy for cancer.⁶⁸ Specifically, dendritic cell-based vaccines are a major type of vaccine in cancer immunotherapy.⁶⁸ PDAC can induce dendritic cell dysfunction, which undermines their ability to introduce neoantigens of tumor cells to T cells, thereby providing a safe microenvironment for cancer cell proliferation.⁶⁹ Consistently, it has been illuminated that restoration of dendritic cell function can markedly potentiate T cell-mediated cytotoxicity in PDAC and improve the efficacy of radiation therapy.⁶⁹

It has been observed that compared with normal dendritic cells, dendritic cells treated with exosomes from PDAC have decreased capacity to activate autologous CD4⁺ and CD8⁺ T cells.⁷⁰ Treatment with exosomes from PDAC differentially upregulated 1,815 lncRNAs and downregulated 1,412 lncRNAs in dendritic cells, suggesting the complex effect of PDAC cells on dendritic cells through ncRNAs.⁷⁰

Interestingly, Que et al. measured the competency of dendritic cells after exposure to lipopolysaccharide (LPS), PDAC-derived exosomes, and ultrafiltered PDAC-derived exosome lysates. After ultrafiltration, PDAC cells lost their miRs, while 150 exosomal proteins were preserved.⁷¹ They showed that PDAC-derived exosomes were inferior to LPS in activating dendritic cells and inducing cytokine-induced killer cell proliferation, cytotoxicity, and TNF-α and perforin secretion against PDAC cells.⁷¹ However, ultrafiltration and subsequent removal of exosomal miRs markedly increased cytokine-induced killer cell proliferation, cytotoxicity, and TNF-α and perforin secretion against PDAC cells compared with PDAC-derived exosomes and LPS.⁷¹ These findings indicate that by releasing exosomal ncRNAs PDAC cells can prevent dendritic cell activation and subsequently hinder the cytolytic function of effector cells.⁷¹

Zhou et al. reported that miR-203 in PDAC-derived exosomes can downregulate TLR4 in dendritic cells and subsequently reduce TNF-α and IL12 release by dendritic cells.⁷² Consistently, miR-203 inhibition upregulated TLR4 expression and enhanced TNF-α and IL12 secretion by dendritic cells.⁷² TLR4 recognizes damage-associated molecular patterns (DAMPs) released by cancer cells and plays a pivotal role in the initial response of the immune system.⁷³ It has been elucidated that the supernatants of chemically stressed cancer cells cannot induce phenotypic maturation and inflammatory cytokine production in TLR4-deficient dendritic cells.⁷³ Consistently, TLR4-deficient dendritic cells could not activate Th1 in response to the supernatants of chemically stressed cancer cells.⁷³

Similar to Zhou et al., Du et al. indicated that medium conditioned by a highly metastatic human pancreatic cancer cell line BxPC-3 markedly inhibited phenotypic differentiation and maturation of dendritic cells, evidenced by decreased expression of CD80, CD86, CD1a, HLA-DR, CD83.⁷⁴ They found that miR-146a was aberrantly upregulated in BxPC-3-conditioned medium, and miR-146a inhibition considerably reversed the inhibitory effects of BxPC-3-conditioned medium on phenotypic differentiation and maturation of dendritic cells.⁷⁴ They also observed that miR-146a inhibition reverses the suppressive effect of BxPC-3-conditioned medium on dendritic cell-mediated activation of cytotoxic T lymphocytes.⁷⁴

Ding et al. revealed that PDAC cells markedly overexpress miR-212–3p, which downregulated regulatory factor X-associated protein (RFXAP), an important transcription factor for MHC II in dendritic cells.⁷⁵ They found that exosomes from three pancreatic cancer cell lines, namely PANC-1, SW1990, and BxPC-3, considerably overexpressed miR-212–3p and markedly downregulated RFXAP and MHC II in dendritic cells.⁷⁵ miR-212–3p inhibition markedly upregulated RFXAP and MHC II, while miR-212–3p mimic significantly decreased the expression of RFXAP and MHC II in dendritic cells treated with PDAC cell exosomes.^75,76 Intriguingly, it has been shown that by inhibiting miR-212–3p in PDAC cell line PANC-1, INF-γ can upregulate RFXAP and MHC II and enhance cancer cell recognition and promote the anticancer response of immune cells.⁷⁶ Similarly, high expression of miR-128 in PDAC cells was shown to promote the expression of MHC I, MHC II, and costimulatory molecules, CD80 and CD86, in dendritic cells cocultured with PDAC cells.⁷⁷ Mechanistically, it was observed that miR-128 downregulates the expression of zinc finger E-box binding homeobox 1 (ZEB1), a transcription factor involved in tumor invasion and progression, thereby leading to the above-mentioned changes in the expression of MHC I, MHC II, CD80, and CD86.⁷⁷ Consistently, it has been found that high expression of miR-128 prognosticates prolonged survival in PDAC.⁷⁷ These complex mechanisms remind the importance of ncRNAs in PDAC cell recognition and regulation of anticancer immune response by dendritic cells (Figure 1).

Figure 1.

The role of ncRNAs in the immunosuppressive effects of PDAC cells on dendritic cells and T cells. PDAC cells upregulate several immunosuppressive ncRNAs (shown by red color) and downregulate immunogenic ncRNAs (shown by green color) to escape recognition by dendritic cells and dampen T cell cytotoxicity. For instance, by altering the expression pattern of ncRNA, PDAC cells can downregulate MHC I, MHC II, CD80/CD86, and TLR4 and hinder identification and antigen presentation by dendritic cells. Similarly, with the help of ncRNAs, PDAC cells can promote PD-1/PD-L1 and FAS/FASL interaction and accelerate T cell death, inhibit the production of inflammatory cytokines or cytotoxic enzymes in T cells, or prevent cancer cell recognition by impairing TCR synthesis.

Although these findings imply the pivotal role of ncRNAs in dendritic cell dysfunction in PDAC, the involvement of a greater number of ncRNAs remains to be defined by future studies. These findings can help attenuate the suppressive effects of PDAC on dendritic cells through ncRNAs, and design more effective immunotherapy regimens (Table 1).

Table 1. ncRNAs Involved in the Immune Evasion of PDAC with Their Corresponding Function.

ncRNA	expression in PDAC	origin	target cells	function	reference
miR-203	upregulated	PDAC cells	dendritic cells	It can downregulate TLR4 in dendritic cells and subsequently reduce TNF-α and IL12 release by dendritic cells.	(72)
miR-146a	upregulated	PDAC cells	dendritic cells	It inhibited phenotypic differentiation and maturation of dendritic cells by decreasing the expression of CD80, CD86, CD1a, HLA-DR, and CD83.	(74)
miR-212-3p	upregulated	PDAC cells	dendritic cells	By downregulating RFXAP, it decreases MHC II transcription in dendritic cells.	(75)
miR-128	downregulated	PDAC cells	dendritic cells, PDAC cells	It enhances the expression of MHC I, MHC II, and costimulatory molecules, CD80 and CD86, in dendritic cells. It also downregulates CD47 in PDAC cells by targeting ZEB1, thereby attenuating the defense mechanism of tumor cells against recognition by immune cells.	(77)
miR-183-5p	NA	PDAC cells	macrophages	It induces the M1 polarization of macrophages.	(80)
miR-301a	upregulated	PDAC cells	macrophages	By attenuating the inhibitory effect of PTEN on PI3K/Akt/mTORC1 pathway, it promotes M2 polarization of macrophages.	(53)
miRNA-320a	upregulated	CAFs	macrophages	By attenuating the inhibitory effect of PTEN on the PI3K/AKT/mTORC1 pathway, it promotes M2 polarization of macrophages.	(33)
miR-340	downregulated	PDAC cells	PDAC cells	It downregulates CD47 on cancer cells and inhibits CD47-SIRP-α interaction, thereby accelerating tumor cell phagocytosis.	(84)
miR-125b	NA		M2 macrophages	It reprograms M2 macrophages to show M1 features.	(86)
miR-21-5p	upregulated	M2 macrophage	PDAC cells	It inhibits KLF3 in PDAC cells, thereby promoting their proliferation.	(82)
miR-501-3p	upregulated	M2 macrophage	PDAC cells	Targeting the 3′-UTR of TGFβR3 in cancer cells and promoting their EMT and angiogenesis.	(83)
miR-122-5p	downregulated	PDAC cells	PDAC cells	By inhibiting XIAP, it induces apoptosis in PDAC cells.	(89)
lncRNA SBF2-AS1	upregulated	M2 macrophage	PDAC cells	It attenuates the inhibitory effect of miR-122-5p on XIAP, thereby preventing PDAC cell apoptosis.	(89)
miR-365	upregulated	M2 macrophage	PDAC cells	It induces resistance to gemcitabine in PDAC cells.	(91)
miR-222-3p	upregulated	M2 macrophage	PDAC cells	It induces resistance to gemcitabine in PDAC cells.	(92)
miR-153	downregulated	PDAC cells	PDAC cells	It targets the 3′-UTR of HIF-1α and ADAM10 in PDAC cells, thereby upregulating MICA and MICB in PDAC cells and NKG2D in neighboring NK cells.	(21)
circ_0000977	upregulated	PDAC cells	PDAC cells	By inhibiting miR-153, it downregulates MICA and MICB in PDAC cells and NKG2D in neighboring NK cells.	(21)
miR-4299	downregulated	PDAC cells	PDAC cells	It markedly downregulates ADAM17 in PDAC cells and upregulates NKG2D in NK cells.	(94)
miR-3607-3p	downregulated	NK cells	PDAC cells	By targeting IL26, it suppressed the proliferation, migration, and invasion of cancer cells.	(99)
lncRNA AL137789.1	upregulated	PDAC cells	CD8+ cells	It inhibits CD8+ cytotoxicity against PDAC cells.	(27)
miR-148a-3p	downregulated	PDAC cells	PDAC cells	It increases the abundance of CD8+ T cells and CD4+ T cells and decreases the abundance of T Reg cells by downregulating PD-L1.	(106)
hsa_circ_0046523	upregulated	PDAC cells	PDAC cells	By inhibiting miR-148a-3p, it upregulates PD-L1, thereby decreasing the abundance of CD8+ T cells and CD4+ T cells and increasing the abundance of T Reg.	(106)
miR-142-5p	NA	PDAC cells	PDAC cells	By targeting the 3′-UTR of PD-L1, it promotes tissue infiltration of CD4+ and CD8+ T cells, reduces the expression of PD-L1 and the population of PD-1-positive tumor-infiltrating lymphocytes, and enhances the expression of INF-γ and TNF-α	(107)
miR-519	downregulated	PDAC cells	PDAC cells	By targeting the 3′-UTR of PD-L1, it promotes T cell cytotoxicity against cancer cells.	(109)
miR-194-5p	downregulated	PDAC cells	PDAC cells	It promotes CD8+ T cell infiltration and increases the release of IL2, INF-γ, and granzyme B by T cells by targeting the 3′-UTR of PD-L1 and decreasing its expression in cancer cells.	(108)
miR-155-5p	upregulated	PDAC cells	PDAC cells	It upregulates immune checkpoint proteins such as FAS and PD-L1, thereby decreasing the tissue abundance of T cells and increasing the tissue abundance of M2 macrophages and MDSCs. It also upregulates CD47.	(110)
miR-503-5p	downregulated	PDAC cells	PDAC cells	It inhibits anilin expression, thereby improving CD8+ T cell response and promotes the M1 polarization of macrophages.	(112)
lncRNA LINC00460	upregulated	PDAC cells	PDAC cells	By sponging miR-503-5p, it suppresses CD8+ T cell response and promoting M2 polarization of macrophages.	(112)
miR-195-5p	downregulated	PDAC cells	PDAC cells	By targeting the 3′-UTR of PD-L1, it enhances CD8+ T cell response.	(117)
LINC00473	upregulated	PDAC cells	PDAC cells	It inhibits miR-195-5p and suppresses CD8+ T cell response.	(117)
let-7c-5p	NA	PDAC cells	CD8+ cells	It suppresses IL2 and INF-γ production by T cells.	(118)
miR-382-3p	downregulated	PDAC cells	PDAC cells	By sponging STAT-1, it inhibits PD-L1 expression and promotes CD8+ cytotoxicity.	(22)
lncRNA PSMB8-AS1	upregulated	PDAC cells	PDAC cells	It inhibits miR-382-3p, thereby upregulating PD-L1 expression and promoting CD8+ cytotoxicity.	(22)
circPTPN22	upregulated	PDAC cells	PDAC cells	By upregulating STAT-3, it hinders tissue infiltration of CD8+ T cells, CD4+ T cell, γδ T cell, and NK cell, and reduces INF-γ and granzyme B production by CD8+ T cells	(119)
circMYO1C	upregulated	PDAC cells	PDAC cells	It upregulates PD-L1 and weakens CD8+ cytotoxicity.	(124)

PDAC Cells Reshape Macrophage Behavior through ncRNAs

The phenotypic maturation of macrophages can determine their competency for removing cancer cells or promoting tumor progression.⁷⁸ PDACs possess an inherent capability for promoting the M2 polarization of macrophages.⁷⁸ Despite the antitumor nature of M1 macrophages, M2 macrophages, also known as tumor-associated macrophages (TAMs), profoundly contribute to PDAC progression by inducing immunosuppression in the tumor microenvironment and accelerating angiogenesis, matrix remodeling, and cancer cell invasion.⁷⁸ Specifically, TAMs have been implicated in cancer cell resistance to immunotherapy.⁷⁹ Previously, it has been reported that pancreatic acinar cells can release exosomal ncRNAs that affect the behavior of adjacent macrophages.⁸⁰ For instance, pancreatic acinar cells induced the M1 polarization of macrophages by releasing exosomal miR-183–5p in a rat model of acute pancreatitis.⁸⁰ Mechanistically, miR-183–5p induced M1 polarization of macrophages by downregulating forkhead box protein O1 (FoxO1), a transcription factor shifting macrophage polarization toward the M2 phenotype.⁸⁰ Recently, it has been demonstrated that PDAC cells employ ncRNAs to target macrophage polarization and function.⁸¹ On the other hand, macrophages, particularly TAMs, recruit ncRNAs to regulate tumor progression in PDAC (Figure 2).^82,83

Figure 2.

The role of ncRNAs in the interaction between PDAC cells and macrophages or NK cells. ncRNAs are deeply involved in the mutual interaction between PDAC cells and macrophages or NK cells. Upregulation of immunosuppressive ncRNAs (shown by red color) and downregulation of immunogenic ncRNAs (shown by green color) by PDAC cells can modulate signaling pathways in naïve macrophages and promote M2 polarization. In return, M2 macrophage-derived ncRNAs can vigorously promote PDAC growth, chemoresistance, invasion, and metastasis, while inhibiting cancer cell apoptosis. On the other hand, ncRNAs that can potentiate NK cell-mediated cytotoxicity are downregulated in PDAC.

Wang et al. reported that patients with pancreatic cancer have significantly higher serum levels of miR-301a.⁵³ They also indicated that high serum levels of miR-301a are associated with the advanced stage of the disease, lymph node metastasis, increased depth of invasion, and shorter survival.⁵³ Mechanistically, they uncovered that in response to hypoxia, PDAC cells upregulate hypoxia-inducible factor (HIF)-1α and HIF-2α, which subsequently promote the expression of miR-301a.⁵³ Consistently, PDAC cells could not overexpress miR-301a in normoxic condition or after HIF-1α and HIF-2α knockdown.⁵³ Exosomal miR-301a released by PDAC cells shifts macrophage polarization toward the M2 phenotype by attenuating the inhibitory effect of PTEN on phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin complex 1 (mTORC1) pathway.⁵³ Intriguingly, they found that miR-301a-treated macrophages enhanced the EMT of PDAC cells by decreasing the expression of E-cadherin and increasing the expression of N-cadherin, vimentin, and matrix metalloproteinase 7 (MMP7).⁵³ Zhao et al. extracted CAFs from patients with PDAC and indicated that CAF-derived exosomal miRNA-320a can induce the M2 phenotype of cocultured macrophage, evidenced by increased expression of IL10, CD163, and CD206.³³ Mechanistically, miRNA-320a targeted the 3′UTR of PTEN and activated the PI3K/AKT/mTORC1 pathway to promote M2 polarization of macrophages and upregulate IL10, CD163, and CD206.³³ These studies elaborately illuminated how ncRNAs are involved in cancer cell-immune cell cross-talk.

Xi et al. demonstrated that low expression of miR-340 or high expression of CD47 predicts shorter survival of PDAC patients and larger size of the tumor in the mice model.⁸⁴ They found that miR-340 directly inhibits CD47 expression in PDAC cells, thereby attenuating the “self” signal delivered by the interaction of CD47 on tumor cells and signal regulatory protein α (SIRP-α) on M1 macrophages.^84,85 By suppressing CD47 expression, miR-340 pronouncedly enhanced tumor cell phagocytosis by M1 macrophages in vitro and increased the abundance of M1 macrophages and CD8+ T cells in PDAC tissue in a mice model.⁸⁴ In addition, macrophage depletion markedly attenuated the inhibitory effect of miR-340 on tumor growth in vivo.⁸⁴

Amiji et al. produced TAM-targeted hyaluronic acid polyethylene glycol/polyethylenimine nanoparticles (HA-PEG/PEI) carrying miR-125b.⁸⁶ They indicated that miR-125b encapsulated by HA-PEG/PEI nanoparticles was highly taken by TAMs and promoted M1 characteristics, evidenced by increased expression of inducible nitric oxide synthase (iNOS) and decreased expression of arginase 1 (Arg1).⁸⁶ Interestingly, miR-125b encapsulated by HA-PEG/PEI nanoparticles and delivered intraperitoneally was highly taken by TAMs in mice bearing PDAC and led to 4-fold increase in the M1-to-M2 macrophage ratio.⁸⁶ Specifically, high uptake of miR-125b by TAMs after encapsulation by HA-PEG/PEI nanoparticles enhanced the expression of iNOS and costimulatory molecules, such as CD80 and CD86, and downregulated M2 markers such as CD163, CD206, and Arg1 compared with HA- scrambled miR encapsulated by PEG/PEI nanoparticles.⁸⁶ In another study, Amiji et al. used HA-PEG/PEI nanoparticles to transfect Panc-1 cells with miR-125b-2-expressing plasmids.⁸⁷ They observed that M2 macrophages cocultured with these PDAC cells or exosomes were reprogrammed to obtain M1 features, evidenced by higher expression of iNOS and IL1β and lower amounts of Arg1.⁸⁷ These findings illuminate that ncRNAs can repolarize macrophages in PDAC, and nanoformulation methods can be applied to overcome the traditional issues in the targeted delivery of ncRNAs.⁸⁶

On the other hand, TAM-derived ncRNAs are heavily involved in PDAC progression.⁸² Herein, Chang et al. revealed that M2 macrophages highly express miR-21–5p. M2 macrophage-derived exosomal miR-21–5p promoted PDAC growth in a mice model.⁸² They observed that miR-21–5p targets Krüppel-like factor 3 (KLF3), a zinc finger transcription factor, in PDAC cells.⁸² Previously, it has been reported that KLF3 inhibition promotes PDAC cell proliferation and prevents their apoptosis.⁸⁸ Similarly, Zhou et al. unveiled that M2 macrophages highly express lncRNA SBF2-AS1, and macrophage exosomal lncRNA SBF2-AS1 can inhibit miR-122–5p in PDAC cells, thereby upregulating X-linked inhibitor of apoptosis protein (XIAP) and preventing apoptosis.⁸⁹ Consistently, lncRNA SBF2-AS1 silencing in M2 macrophage exosomes upregulated miR-122–5p expression, downregulated XIAP expression in PDAC, and reduced tumor growth in a mice model.⁸⁹

Zhang et al. indicated that M2 macrophages in the PDAC microenvironment release a high amount of exosomal miR-501–3p, which promotes cancer cell proliferation, invasion, angiogenesis, and metastasis and prevents apoptosis.⁸³ Mechanistically, macrophage-derived exosomal miR-501–3p targeted the 3′-UTR of transforming growth factor beta receptor 3 (TGFβR3), a tumor suppressor, in PDAC cells and subsequently enhanced TGFβR1 and TGFβR2 expression and Smad3 phosphorylation and reduced E-cadherin expression to facilitate epithelial-mesenchymal transition (EMT) and invasion of cancer cells.⁸³ By targeting the 3′-UTR of TGFβR3, miR-501–3p also upregulated angiogenic factors such as vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor receptor 2 (VEGFR2), and angiogenin both in vivo and in vitro.⁸³ Consistently, inhibition of miR-501–3p by antagomiR-501–3p reduced tumor growth, decreased the number of lung and liver nodules, and partly attenuated the effect of M2 macrophage-derived exosomes on PDAC growth in a mice model.⁸³ In line with these findings, previous studies have shown that high expression of TGFβR3 markedly inhibits angiogenesis, migration, invasion, and lymph node metastasis in head and neck cancer.⁹⁰

Likewise, it was found that TAMs can release a high amount of exosomal miR-365 that can enhance the resistance of the K989 PDAC cell line to gemcitabine.⁹¹ Consistently, K989 cells or mice treated with antago miR-365 had a better response to gemcitabine, showing that ncRNAs released by TAMs may induce chemoresistance in PDACs.⁹¹ Besides, it was shown that M2 macrophage-derived exosomes are enriched in miR-222–3p, which was shown to similarly promote the resistance of PDAC to gemcitabine, prevent cancer cell apoptosis, and enhance cancer cell proliferation both in vivo and in vitro.⁹² Consistently, miR-222–3p inhibitor partly reversed the effect of M2 macrophage-derived exosomes on the resistance of PDAC to gemcitabine in a mice model. Mechanistically, miR-222–3p inhibited tuberous sclerosis 1 (TSC1), a tumor suppressor, thereby upregulating the proliferative pathway of PI3K/AKT/mTORC1 in cancer cells.⁹²

This section exemplified the role of ncRNAs in PDAC cell-macrophage interaction and discussed several therapeutic targets for the immunotherapy of PDAC (Figure 2 and Table 1). Future studies can discover much more of these relationships and expand our knowledge of the role of ncRNAs in the immunotherapy of PDAC.

The Involvement of ncRNAs in NK Cell Function in PDAC

NK cells are responsible for a major proportion of the anticancer response of innate immunity as they have direct cytotoxic activity against cancer cells similar to T cells.⁵⁴ Recent studies have shown that patients with PDAC not only exhibit decreased intratumor NK cell population but also their NK cells are functionally less-competent, indicated by low expression of CD16, CD57, DNAM-1, NKP30, IFN-γ, and other markers of NK cell activation.⁵⁴ Additionally, NK cells and their exosomes are crucial for the competent function of T cells.⁹³ Therefore, NK cell-targeted therapy has been proposed as a potential method of immunotherapy in PDAC.

ncRNAs have newly emerged as major regulators of NK cell function in PDAC.^21,94 Herein, Ou et al. revealed that in response to hypoxia, PDAC cells upregulate circ_0000977 to sponge miR-153 and attenuate its inhibitory effects on HIF-1α and A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10).²¹ They found that miR-153 targets the 3′-UTR of HIF-1α and ADAM10 in PDAC cells and shortens cancer cell viability in hypoxia.²¹ By upregulating HIF-1α in PDAC cells, circ_0000977 lowered the expression of MICA and MICB in PDAC cells and NKG2D in cocultured NK cells, increased the expression of soluble MICA (sMICA) in culture media, and blunted the cytotoxic function of cocultured NK cells.²¹ Consistently, miR-153 mimic or circ_0000977 inhibition markedly suppressed HIF-1α expression and upregulated the expression of NKG2D in NK cells and MICA and MICB in PDAC cells. They also increased the expression of ADAM10 in PDAC cells and downregulated sMICA in culture media, and reinforced the cytotoxic function of cocultured NK cells.²¹ Likewise, it was observed that miR-4299 is lowly expressed in pancreatic cancer tissues and cell lines.⁹⁴ In addition, miR-4299 overexpression markedly reduced PDAC cell proliferation in vitro and in vivo by targeting the 3′UTR of ADAM17.⁹⁴ Moreover, miR-4299 enhanced the NKG2D-positive rate and killing ability of NK cells.⁹⁴ ADAMs, generically known as sheddases, cleave membrane proteins such as MICA and MICB at the cellular surface and have been implicated in cancer cell immune evasion.^95,96 NK cells can recognize and target cells via the NKG2D receptor.⁹⁷ They can identify and destroy tumor cells by recognizing MICA and MICB on tumor cell surface.⁹⁷ Tumor cells proteolytically cleave MICA and MICB by surface proteases and shed them into the extracellular space to evade NKG2D recognition.⁹⁷ Therefore, low expression of membrane MICA and MICB and high expression of soluble MICA and MICB are common findings in cancer tissue.⁹⁷ By upregulating membrane-bound MICA and MICB, miR-4299 can promote NK cell function and stimulate the expression of NKG2D, a surface receptor of NK cells for membrane-bound MICA and MICB. Moreover, Chen et al. revealed that compared with adjacent nontumor tissues, NKG2D is significantly downregulated and sMICA is markedly upregulated in PDAC tissue.⁹⁸ They also indicated that despite sMICA, high expression of NKG2D is associated with prolonged disease-free survival and overall survival.⁹⁸ Moreover, multivariate regression uncovered that high expression of sMICA in PDAC is an independent predictive factor for shorter disease-free survival and overall survival.⁹⁸ These findings imply that by means of ncRNAs, PDAC cells can evade NK cell response (Figure 2 and Table 1).

On the other hand, NK cell-derived exosomal miR-3607–3p was shown to hinder the proliferation, migration, and invasion of PaCa-2 and Panc-1 cell lines in vitro.⁹⁹ It was also found that the plasma levels of exosomal miR-3607–3p and tissue levels of miR-3607–3p were lower in patients with PDAC compared with normal individuals.⁹⁹ Furthermore, low levels of plasma exosomal miR-3607–3p were associated with lymph node metastasis and shorter survival among patients with PDAC.⁹⁹ Consistently, miR-3607–3p inhibition significantly abolished the inhibitory effect of NK cell-derived exosomes on PDAC growth and invasion.⁹⁹ Mechanistically, miR-3607–3p targeted IL26 in PDAC cells.⁹⁹ The study revealed that the expression of IL26 was higher in PDAC tissues than in normal tissues, and it increased with lymph node metastasis.⁹⁹ Previous studies illuminated that IL26 production by cancer cells can dampen T cell response and prevent apoptosis in cancer cells.¹⁰⁰

Future studies are needed to identify more ncRNAs in the mutual interaction of PDAC cells and NK cells. ncRNA-based immunotherapy may ameliorate NK cell dysfunction in PDAC (Figure 2 and Table 1).

PDAC Cells Can Evade T Cell Cytotoxicity through ncRNAs

The primary response by innate immune cells, such as NK cells and dendritic cells, summons effector CD8+ T cells to the tumoral tissue and provokes their anticancer function.¹⁰¹ PDAC cells employ various mechanisms to escape T cell cytotoxicity, warranting the limited efficacy of a single ICI or the combination of several ICIs for treating this cancer.¹⁰² PDAC cells recruit different types of suppressive cells such as T Reg cells, CAFs, M2 macrophages, and myeloid-derived suppressive cells (MDSCs) with their anti-inflammatory cytokines to dampen T cell response.¹⁰³ Thus, immunotherapy strategies targeting several mechanisms of immune evasion are deemed to be more effective than those targeting a single mechanism.¹⁰²

RNA-based treatment of PDAC has been widely used in preclinical studies which showed promising results.¹⁰⁴ For instance, Yoo et al. administered the combination of gemcitabine and small interfering RNA against PD-L1 to treat PDAC in a mice model.¹⁰⁴ They found that the combination therapy achieved a 90% reduction in tumor volume within 2 weeks.¹⁰⁴ In addition, 67% of mice treated with combination therapy survived for 12 weeks, whereas all mice treated with the combination of scrambled siRNA and gemcitabine died within 6 weeks.¹⁰⁴

Wang et al. reported that lncRNA AL137789.1 is markedly upregulated in human PDAC tissue. High expression of lncRNA AL137789.1 prognosticated worse overall survival of patients and was associated with higher invasion rates of cancer cells.²⁷ Further experiments revealed that lncRNA AL137789.1 overexpression significantly inhibits CD8+ cytotoxicity against BxPC3 and PANC1 cells, whereas lncRNA AL137789.1 silencing potentiates CD8+ cytotoxicity against PDAC cell lines.²⁷ Similarly, Jung et al. developed siPD-L1@poly lactic-co-glycolic acid (PLGA), which significantly suppressed PDAC growth in a mice model and improved the cytotoxic function of T cells.¹⁰⁵

hsa_circ_0046523 was shown to be upregulated in PDAC tissues, and its overexpression predicted aggressive behavior of PDAC cells and shorter survival of patients.¹⁰⁶ It was found that forced expression of hsa_circ_0046523 markedly decreased the abundance of CD8+ T cells and CD4+ T cells and increased the abundance of T Reg cells in peripheral blood mononuclear cells cocultured with PDAC cells.¹⁰⁶ Consistently, hsa_circ_0046523 silencing significantly increased CD8+ and CD4+ population and reduced T Reg cell population.¹⁰⁶ In addition, the study showed that forced expression of hsa_circ_0046523 promoted the expression of anti-inflammatory cytokines such as TGF-β and IL10 and downregulated pro-inflammatory cytokines such as INF-γ and IL2, whereas hsa_circ_0046523 silencing reversed these effects.¹⁰⁶ Mechanistically, hsa_circ_0046523 sponged miR-148a-3p and attenuated its inhibitory effect on PD-L1 expression.¹⁰⁶ Similar to miR-148a-3p, miR-142–5p, miR-194–5p, miR-519, and miR-128 prevented tumor growth and potentiated immune infiltration in a mice model of PDAC.^107−109 Herein, it was shown that miR-142–5p targets the 3′-UTR of PD-L1 in PDAC, promotes tissue infiltration of CD4+ and CD8+ T cells, reduces the expression of PD-L1 and the population of PD-1-positive tumor-infiltrating lymphocytes, and enhances the expression of INF-γ and TNF-α.¹⁰⁷ Similarly, increasing the decreased expression of miR-194–5p in a mice model of PDAC promoted CD8+ T cell infiltration, increased the release of IL2, INF-γ, and granzyme B by T cells, and led to tumor shrinkage by targeting the 3′-UTR of PD-L1 and decreasing its expression in cancer cells.¹⁰⁸ Interestingly, it was shown that in hypoxic conditions, PDAC cells downregulate miR-519 and attenuate its inhibitory effect on PD-L1 expression.¹⁰⁹ On the contrary, PDAC cells can release a high amount of exosomal miR-155–5p, which acts as a defense mechanism and leads to T cell depletion.¹¹⁰ In addition, by releasing miR-155–5p, PDAC cells can induce M2 polarization of macrophages and markedly increase M2/M1 ratio, which provides an immunosuppressive microenvironment for cancer growth.¹¹⁰ Inhibition of miR-155–5p by antagomiR-155–5p considerably increased the tissue abundance of natural killer T cells, decreased the tissue abundance of M2 macrophages and MDSCs, and downregulated checkpoint proteins such as FAS and PD-L1 in a rat model of PDAC. Mechanistically, antagomiR-155–5p inhibited miR-155–5p-mediated activation of the Akt/NF-κB signaling pathway.¹¹⁰ It seems that PDAC cell modulate the expression of ncRNAs in harsh conditions to defend themselves against the immune response (Figure 1 and Table 1).

Treatment with miR-128 significantly increased the intratumor abundance of CD8+ T cells, dendritic cells, and natural killer T cells in a mice model of PDAC and reinforced the phagocytosis capacity of macrophages against cocultured PDAC cells.⁷⁷ Further experiments revealed that miR-128 can suppress the immunosuppressive pathway ZEB1/CD47, thereby unleashing a vigorous anticancer response against PDAC cells.⁷⁷ On the contrary, it has been observed that by overexpressing miR-155–5p, PDAC cells can enhance the expression of CD47 in the tumor microenvironment.¹¹⁰ Previously, it has been uncovered that CD47 expression is positively correlated with the expression of many immune checkpoints in cancer, and CD47 overexpression predicts shorter survival in patients with cancer.¹¹¹ As a signaling molecule inducing immunosuppression, CD47 expression is of great importance in PDAC cells. By regulating CD47 expression in tumor cells, ncRNAs can control the activation of a series of immune cells, modulate their gene expression patterns, and affect how immune cells present their surface receptors and cytotoxic mediators.

Yao et al. showed that compared with adjacent tissues, lncRNA LINC00460 is highly expressed in PDAC tissue and its expression continues to increase with the WHO stage of PDAC or with lymph node metastasis.¹¹² Similarly, the expression of lncRNA LINC00460 was markedly higher in several PDAC cell lines such as ASPC-1, BxPC-3, SW1990, Panc-1, and Mia-PaCa-2 compared with normal human pancreatic duct epithelial cell line (H6C7 cells).¹¹² Furthermore, high expression of lncRNA LINC00460 in the tumor predicted shorter overall survival of patients with PDAC compared with its low expression.¹¹² Mechanistically, by inhibiting miR-503–5p, lncRNA LINC00460 blunts CD8+ response, upregulates M2 macrophage markers such as CD163, CD206, IL10, and Arg1, and downregulates M1 markers such as IL12 and iNOS.¹¹² Mechanistically, the study indicated that miR-503–5p targets anilin in PDAC cells.¹¹² In contrast, treatment with sh-LINC00460 markedly decreased tumor growth both in vivo and in vitro, enhanced CD8+ cytotoxicity, and potentiated the inhibitory effect of anti-PD-1 therapy on PDAC in a mice model.¹¹² Previously, it has been unfolded that anilin acts as an oncoprotein in PDAC and promotes PDAC development, migration, and invasion.¹¹³ In addition, previous studies found that anilin is overexpressed in several types of cancer, and its expression is associated with the expression of immune checkpoints and tumor immunity.¹¹⁴ Arg1 degrades l-arginine, an amino acid required for the biosynthesis of T cell receptor (TCR)-associated CD3ζ chain.^115,116 Therefore, high expression of Arg1 in the tumor microenvironment can downregulate TCR, impair signal transduction in T cells, and induce an immunosuppressive condition.^115,116

Zhou et al. indicated that lncRNA LINC00473 is upregulated in PDAC.¹¹⁷ It promotes the expression PD-L1 in this cancer and suppresses CD8+ T cell response by inhibiting miR-195–5p.¹¹⁷ miR-195–5p specifically targets the 3′-UTR of PD-L1.¹¹⁷ Interestingly, miR-195–5p inhibition attenuated the inhibitory effect of atezolizumab, a PD-L1 inhibitor, on the invasion of PDAC cells.¹¹⁷

Li et al. reported that IL-36β increased the abundance of CD8⁺ T and NK cells in a mice model of PDAC, promoted IL2 and INF-γ production by T cells, and decreased tumor growth, mainly through downregulation of let-7c-5p.¹¹⁸ In addition, upregulation of let-7c-5p in CD8+ T cells undermined their ability to produce IL2 and INF- γ and attenuated the immunotherapeutic effect of IL-36β, while downregulation of let-7c-5p in CD8+ T cells markedly enhanced IL2 and INF- γ release.¹¹⁸ These findings indicate that ncRNAs not only mediate resistance to immunotherapy in PDAC but also provide an opportunity to improve the efficacy of immunotherapy in PDAC.^117,118

Zhang et al. indicated that lncRNA PSMB8-AS1 is markedly overexpressed in PDAC tissues and its high expression foretells their shorter survival.²² Furthermore, lncRNA PSMB8-AS1 was shown to promote PDCA cell proliferation in vitro and PDCA growth and metastasis in a mice model.²² Mechanistically, the study unveiled that lncRNA PSMB8-AS1 sponges miR-382–3p and abolishes the inhibitory of miR-382–3p effect on the expression of STAT-1, a major transcription factor for checkpoint proteins.²² Further experiments unfolded that by upregulating the expression of STAT-1, lncRNA PSMB8-AS1 can promote the expression of PD-L1 in PDAC cells, induce the apoptosis of CD8+ T cells, and reduce the percentage of activated CD8+ T cells in PDAC.²² Consistently, STAT-1 inhibition by sh-STAT-1 reversed the immunosuppressive effects of lncRNA PSMB8-AS1 in PDAC.²² Moreover, a positive correlation was found between lncRNA PSMB8-AS1 and STAT-1 or PD-L1 in PDAC tissues from xenografts or patients with PDAC.²² Similar to lncRNA PSMB8-AS1, circPTPN22 is highly expressed in PDAC, promotes PDAC growth, and induces tolerance in the tumor microenvironment partly through STAT-3 activation and acetylation.¹¹⁹ In particular, it was shown that circPTPN22 enhances STAT-3 activation and acetylation by inhibiting SIRT1-mediated deacetylation of STAT3.¹¹⁹ Using a mice model of PDAC, it was shown that circPTPN22 knockdown by sh-circPTPN22 markedly prevents tumor growth, promotes tissue infiltration of CD8+ T cells, CD4+ T cell, γδ T cell, and NK cell, and reinforces INF-γ and granzyme B production by CD8+ T cells.¹¹⁹ Previously, it has been unfolded that in response to activated T cells, PDAC cells upregulate the oncogenic Janus kinase (JAK)/STAT signaling pathway, thereby augmenting the expression of anti-inflammatory cytokines and immune checkpoint proteins.¹²⁰ In particular, it was observed that INF-γ released by T cells can activate JAK/STAT signaling pathway in cancer cells and subsequently promote PD-L1 expression.¹²⁰ Consistently, inhibition of STAT-1 and STAT-3 by ruxolitinib, a selective STAT inhibitor, markedly enhanced the tissue infiltration of cytotoxic T lymphocytes into PDAC in a mice model.¹²⁰ Interestingly, ruxolitinib was less effective in T-cell-deficient mice, showing the intricate association between the anticancer response of T cells and JAK/STAT signaling pathway.¹²⁰ These findings suggest that PDAC cells overexpress specific ncRNAs to sustain the function JAK/STAT signaling pathway, subsequently inducing tolerance in immune cells and resistance to immune checkpoint blockade.^119,120 Therefore, inhibition of these ncRNAs in combination with other methods of immunotherapy, such as immune checkpoint blockade, may offer a higher rate of success and more effectively prolong patients′ survival (Figure 1 and Table 1).

M6A modification is a type of post-transcriptional epigenetic modification that can regulate RNA stability, translation, and splicing.¹²¹ Newly, it has been demonstrated that m6A mRNA modification by methyltransferase 3 (METTL3) plays a major role in the progression of PDAC.¹²² METTL3 is highly expressed in PDAC and upregulates the expression of lncRNA MALAT1 in PDAC cells.^122,123 In return, lncRNA MALAT1 upregulates PD-L1 expression in PDAC cells and protects cancer cells against T cell response.¹²³ Similarly, it was illuminated that METTL3 enhances the expression and circularization of circMYO1C in PDAC cells.¹²⁴ Subsequently, circMYO1C promotes the mRNA stability and protein expression of PD-L1 in PDAC.¹²⁴ Consistently, circMYO1C knockdown inhibited PDAC growth in a mice model.¹²⁴

However, previous studies elaborately indicated how ncRNAs can modulate cancer cell-T cell interaction, most of them focused on PD-1/PD-L1 interaction and neglected other immune checkpoints and molecular targets. Identification of ncRNAs that target other immune checkpoints and molecular targets in T cell response can help design more effective therapeutic plans for PDAC.

4. Strengths

This narrative review article comprehensively discussed the existing preclinical evidence regarding the potential use of ncRNA in the management of PDAC. Despite previous reviews, this study specifically focused on ncRNAs involved in the immune evasion of only PDAC. In addition, the molecular mechanisms have been thoroughly delineated, which can help identify the potential synergistic effects of current modalities of immunotherapy and ncRNAs and guide the design of future preclinical and clinical studies. In addition, the mechanistic viewpoint provided by this narrative review can help target relevant ncRNAs to overcome the resistance of PDAC to current modalities of immunotherapy.

5. Limitations

Although the role of ncRNAs in PDAC has been mechanistically reviewed in this study, more studies are needed to offer a more comprehensive mechanistic insight and investigate the role of other ncRNAs. In addition, clinical studies proactively adopting ncRNAs to monitor the response to immunotherapy or to overcome resistance to immunotherapy in PDAC are scarce, necessitating future clinical trials in this regard. Furthermore, this study was not a systematic review; thus, some relevant studies might have been missed.

6. Conclusion and Future Prospect

ncRNAs are deeply involved in the regulation of the immune microenvironment in PDAC and play a critical role in cancer cell-immune cell interaction. Through ncRNAs, PDAC cells can suppress the anticancer response of dendritic cells, macrophages, NK cells, and T cells. ncRNAs give PDAC cells the ability to resist chemotherapeutic agents and ICIs. Consistently, preclinical studies that administered the combination of ICIs and ncRNA-based strategies achieved promising results. Therefore, future clinical studies are encouraged to include ncRNA-based strategies in the immunotherapy of PDAC to obtain more favorable results and overcome the shortcoming of ICIs. For instance, many ncRNAs were shown to target PD-L1 (Figure 3). In addition, ncRNA delivery by nanoparticles is an emerging entity that can pave the way for the widespread use of ncRNAs in future studies of PDAC. Besides, more preclinical studies are needed to identify and validate other ncRNAs that can target immune evasion mechanisms that were not addressed by previous studies.

Figure 3.

ncRNAs targeting PD-L1 and T cell cytotoxicity in PDAC.

Glossary

Abbreviations

ADAM10

A disintegrin and metalloproteinase domain-containing protein 10

Arg1

Arginase 1

BRAF

B-Raf proto-oncogene

CAFs

cancer-associated fibroblasts

circRNAs

circular RNAs

CXCL

C-X-C motif chemokine

CXCR3

C-X-C motif chemokine receptor 3

CTLA-4

cytotoxic T lymphocyte-associated antigen 4

DAMPs

damage-associated molecular patterns

DALYs

disability-adjusted life year

DNAM-1

DNAX activation motif-1

EMT

epithelial-mesenchymal transition

FOXP3

forkhead box P3

FoxO1

forkhead box protein O1

HAVCR2

hepatitis A virus cellular receptor 2

HA-PEG/PEI

hyaluronic acid polyethylene glycol/polyethylenimine nanoparticles

HIF

hypoxia-inducible factor

ICIs

immune checkpoint inhibitors

(IDO)1

indoleamine 2,3-dioxygenase

iNOS

inducible nitric oxide synthase

IFN-γ

interferon gamma

KIR3DL1

killer cell immunoglobulin-like receptor 3DL1

KRAS

Kirsten rat sarcoma virus

KLF3

Krüppel-like factor 3

lncRNAs

long noncoding RNAs

LAG-3

lymphocyte activation gene 3

MHC

major histocompatibility complex

MICA/B

MHC class I chain-related molecule A and B

miRs

microRNAs

mTORC1

mammalian target of rapamycin complex 1

MMP7

matrix metalloproteinase 7

MYC

myeloid cell-specific nuclear antigen

MDSCs

myeloid-derived suppressive cells

NK

natural killer

NGAL

neutrophil gelatinase-associated lipocalin

NKP30

NK cell-activating receptor

NKG2D

NK group 2D

ncRNAs

noncoding RNAs

PDAC

pancreatic ductal adenocarcinoma

PTEN

phosphatase and tensin homologue

PI3K

phosphatidylinositol 3-kinase

PD-1

programmed death 1

PD-L1

programmed death ligand 1

PD-L2

programmed death ligand 2

AKT

protein kinase B

RFXAP

regulatory factor X-associated protein

RBP-4

retinol-binding protein-4

RNAs

ribonucleic acids

SIRP-α

signal regulatory protein α

STAT

signal transducer and activator of transcription

sMICA

soluble MICA

TIM3

T cell immunoglobulin and mucin domain-containing protein 3

TNFSF9

TNF superfamily member 9

TNFRSF9

TNF receptor superfamily member 9

TLR4

toll-like receptor 4

TGFβR3

transforming growth factor beta receptor 3

TSC1

tuberous sclerosis 1

TAMs

tumor-associated macrophages

TNF-α

tumor necrosis factor α

VEGFA

vascular endothelial growth factor A

VEGFR2

vascular endothelial growth factor receptor 2

XIAP

X-linked inhibitor of apoptosis protein

ZEB1

zinc finger E-box binding homeobox 1

Author Contributions

M.A. conceptualized this article, performed the literature search, edited the draft, and created the figures.

The author declares no competing financial interest.