Emerging role of chemokines in small cell lung cancer: Road signs for metastasis, heterogeneity, and immune response

Abstract

Small cell lung cancer (SCLC) is a recalcitrant, relatively immune-cold, and deadly subtype of lung cancer. SCLC has been viewed as a single or homogenous disease that includes deletion or inactivation of the two major tumor suppressor genes (TP53 and RB1) as a key hallmark. However, recent sightings suggest the complexity of SCLC tumors that comprises highly dynamic multiple subtypes contributing to high intratumor heterogeneity. Furthermore, the absence of targeted therapies, the understudied tumor immune microenvironment (TIME), and subtype plasticity are also responsible for therapy resistance. Secretory chemokines play a crucial role in immunomodulation by trafficking immune cells to the TIME. Chemokines and cytokines modulate the anti-tumor immune response and wield a pro-/anti-tumorigenic effect on SCLC cells after binding to cognate receptors. In this review, we summarize and highlight recent findings that establish the role of chemokines in SCLC growth and metastasis, and sophisticated intratumor heterogeneity. We also discuss the chemokine networks that are putative targets or modulators for augmenting the anti-tumor immune responses in targeted or chemo-/immuno-therapeutic strategies, and how these combinations may be utilized to conquer SCLC.

1. Introduction

Small cell lung cancer (SCLC) is a highly aggressive lung cancer (LC) subtype that accounts for nearly 13–15% of total LC cases [1, 2]. Early metastasis and acquired therapeutic resistance are the major factors that limit the long-term outcomes of the disease [3]. Initially, SCLC was considered a homogenous disease and based on the extent of spread at diagnosis; it is categorized into limited-stage (LS) SCLC (~30%) or extensive-stage (E.S.) SCLC (~70%). The major characteristics of SCLC include high genomic instability and vascularity, and the genetic landscape of SCLC is very complex; however, the functional inactivation of the two primary tumor suppressor proteins TP53 and retinoblastoma 1 (RB1) is universal [4, 5]. Besides high mutational rate or loss of function mutations in TP53 and RB1, other molecules such as Myc proto-oncogene protein (MYC), nuclear factor IB (NFIB), and NOTCH demonstrated a significant role in SCLC regulation and progression [6–10].

Interestingly, the current advances in the SCLC field confirm the existence of four major subclasses of SCLC, including SCLC-A [high acheate-scute homologue 1 (ASCL1)], SCLC-N [high neurogenic differentiation factor 1 (NEUROD1)], SCLC-Y [high yes associated protein 1 (YAP-1)], and SCLC-P [high POU class 2 homeobox 3 (POU2F3)] [6, 11, 12]. Recently, two more subtypes have been identified; one named as inflamed (I) subtype based on high expression of immune checkpoint molecules with profound immune cells infiltration, whereas the other one has been characterized by high expression of phospholipase C gamma 2 (PLCG2) [13, 14]. These subtypes have low expression of ASCL1, NEUROD1, POU2F3, and YAP1 [13, 14]. The differential expression of subtype-specific markers (ASCL1, NEUROD1, YAP-1, POU2F3, and PLCG2) categorize SCLC a tumor with high heterogeneity, and the subtype switching accompanied the acquired resistance to platinum-based chemotherapies [13–16]. Subtype-I and -P were investigated as immune hot tumors, whereas subtype-A and -N were considered as immunologically cold tumors [13, 15].

Initially, 60–70% of SCLC cases respond well to conventional chemotherapy (cisplatin/etoposide), and LS-SCLC patients are treated with concurrent chemotherapy with or without thoracic and cranial irradiation, which has improved survival outcomes, but the recurrence is almost universal [17, 18]. Due to the lack of treatment options, SCLC was designated as forgotten cancer for a very long time. Recently, lurbinectedin was added to the second-line treatment [19]. In addition, the recent developments in immunotherapy have added new drugs to the SCLC treatment regimen, including immune checkpoint inhibitors (ICI) atezolizumab, and durvalumab [20–24]. The immunotherapy is administered in conjunction with platinum-based chemotherapy, or in some cases, atezolizumab or durvalumab is given as maintenance therapy [22–25]. Although new improvements in immunotherapy have increased the overall survival of SCLC patients, the outcomes are not very promising [14, 26]. This drawback may stem from the defects in the proliferation or entry of tumor-infiltrating lymphocytes (TILs) at the tumor site. Therefore, understanding the tumor immune microenvironment (TIME) in SCLC is crucial for effective treatment.

The TIME was constituted by various components, to which cytokines/chemokines are the major contributors [27, 28]. Chemokines are small, secreted proteins that mediate lymphoid tissue development and immune cell trafficking [27, 28]. They are the major subfamily of cytokines and can be divided into four subfamilies, depending on the first two cysteine residues in their amino acid sequences: C-chemokines, C-C chemokines, CXC-chemokines, and CX₃C-chemokines [28]. In the TIME, both immune and non-immune cells express chemokines and their cognate receptors [29, 30]. Different immune cells regulate the TIME in a spatiotemporal manner in response to specific chemokines [31]. Chemokines can be divided into homeostatic and inflammatory based on their mechanism of action. Chemokines shape the tumor’s immune and biological phenotypes. They have been shown to regulate cancer growth, proliferation, stemness, and metastasis, thereby affecting the overall outcomes of cancer patients [29, 31]. The research on chemokines reached an age, and given the multidimensional role of chemokines and their receptors in multiple cancers, it was established that chemokines provide a potential axis for developing anticancer therapeutic modalities for various cancers, including SCLC. Although the role of chemokines in SCLC is understudied, some interesting recent investigations revealed that chemokines play some exciting roles in SCLC heterogeneities and differential therapeutic responses [13, 14, 32]. In this review, we aim to summarize specifically the role of chemokines in regulating SCLC tumor microenvironment (TME) and emerging evidence provide their subtype-specific role in modulating immune response and metastasis of SCLC. We also discuss if targeting chemokine or chemokine receptor-mediated signaling can boost the efficacy of chemo-/immuno-therapies in SCLC.

2. Chemokines in SCLC

Tumor-promoting inflammation is an essential component of the TME. It is one of the major hallmark capabilities that trigger tumor growth and progression [33]. Initially, chemokines were thought to determine the composition of tumor stroma, but various studies have reported them directly affecting cancer progression and metastasis [28, 29]. Immune cells such as myeloid-derived suppressor cells (MDSCs), T helper 2 (Th2) cells, T-regulatory (Treg) cells, plasmacytoid dendritic cells (pDCs), and innate lymphoid cells (ILCs) can promote tumor growth [29, 34]. Different chemokines expressed in the TME recruit the pro-tumor immune cells, which inhibit the anti-tumor immune response (Fig. 1) [29, 34]. They are also involved in promoting and maintaining cancer stemness and angiogenesis, the critical factors for cancer progression [28].

Figure 1: Cascade of chemokines in SCLC.

Tumor-associated macrophages (TAMs), cancer-associated fibroblasts (CAFs), and SCLC cells secreted various chemokines such as CCL19, CCL21, CCL8, and CXCL12. The receptors (CXCR4 and CCR1) for these chemokines are present in the SCLC cells. CXCL12 released from TAMs/CAFs binds to CXCR4 to enhance SCLC invasiveness and metastasis. TAMs also contribute to pro-tumorigenic response in SCLC progression through the production of CCL17, and CCL22 that attract CCR4+/CCR8+ regulatory T (Treg) cells, whereas SCLC cells secret CCL19 and create an immune-inflamed environment. Further, SCLC cells contribute toward suppressing cytotoxic T and NK cells activities by producing CXCL5, CXCL8, CXCL12, and CCL19. These chemokines also decreased the differentiation or production of immune cells. The CCL5 and CXCL10 chemokines attract CXCR3 and CCR5 positive immune cells and contribute to the recruitment of immune cells in SCLC tumors.

2.1. Chemokines in tumor-associated angiogenesis and metastases

Tumor-associated angiogenesis is one of the essential features that regulate the growth, development, and progression of solid tumors [35]. The TME orchestrates vascular remodeling; as tumor lesion progresses, it is accompanied by hypoxia that induces an angiogenic switch, which facilitates cancer progression [36, 37]. Tumor cells release chemokines and growth factors such as the vascular endothelial growth factors (VEGF), a critical regulator of angiogenesis that leads to endothelial cell sprouting by exerting mitogenic and anti-apoptotic effects [38]. It binds VEGF receptors VEGFR1 and VEGFR2, expressed on the vascular endothelial cells, and enhances the vascular network [38]. The CXC and CC chemokines play a profound role in cancer-related angiogenesis by directly influencing the expression of VEGF or recruiting effector immune cells into the TME, which produces angiogenic factors [39, 40]. These angiogenic factors can work in a direct, serial, or parallel manner to promote vascular networks [38].

Most CXC chemokines have an angiogenic and/or lymphangiogenic role in the TME [32, 41, 42]. In cancer cells, there is an upregulation of the pro-angiogenic CXC-chemokines [37, 42, 43]. Although the direct effect of CXCL17 on tumor cells is not defined, inhibition of CXCL17/CXCR8 axis by miR-325–3p results in downregulation of angiogenesis in hepatocellular carcinoma [44]. CXCL12 induces the expression of VEGF in endothelial cells and monocytes [45]. Similarly, the CXCL12/CXCR4 axis blockade has prevented metastasis in preclinical mouse models [46]. Hypoxia-induced upregulation of CXCL12 has been observed in hematopoietic stem cells, fibroblasts, and various human cancers. This upregulation occurs in a HIF-1α- and HIF-2α- dependent manner [46]. CXCL12 expression in the TME results in the recruitment of IL-17 secreting-Th17 cells into the tumor. IL-17 stimulation increases VEGF levels and promotes angiogenesis. IL-17 also promotes angiogenic factors such as IL-6, IL-8, and VEGF in lung adenocarcinoma mouse models [47, 48]. SCLC cells produced high levels of IL-8 and VEGF that contribute to SCLC metastasis and angiogenesis [49]. CXCL12 also recruits CXCR4-expressing B cells into the TME [50, 51]. B cells have been shown to enhance tumor-associated angiogenesis in vivo through Stat3 activation [51, 52]. CXCL8, also known as IL-8, is a CXC-chemokine that has been extensively studied as an angiogenesis-mediator [53]. CXCL8 expression by tumor cells and myeloid cells recruits neutrophils [53–55]. These tumor-associated neutrophils secrete molecules for promoting angiogenesis in the colon and breast cancer [53]. High expression of CXCR1 and CXCR2 has been reported in SCLC cell lines, and exogenous stimulus of CXCL8 enhanced the proliferation of SCLC cell lines [56]. CXCL8 acts as a prominent angiogenic factor in various cancers, and the high expression of CXCL8 receptors (CXCR1 and CXCR2) suggests that this chemokine has a budding role in SCLC metastasis. Hypoxia-induced CCL28 also promotes angiogenesis in lung adenocarcinomas by targeting CCR3 in the microvascular endothelial cells [57].

SCLC is a disease of systemic metastases, and the critical supporting factor for metastasis is angiogenesis. A recent interesting study demonstrated that CCR1, CCL8, CCL19, and CCL21 were the differentially expressed chemokines in primary lesions versus metastatic lymph nodes of SCLC patients (Table 1) [32]. This study compared chemokines in metastatic lymph nodes and primary tumors of SCLC patients and found that CCL19, CCL21, CCL8, and CCR1 were most differentially expressed chemokines [32]. These chemokines were further tested in multiple SCLC cell lines (H69, H446, H196, H82) and found that CCL19 mRNA and protein expression were both upregulated profoundly as compared to BEAS2B (normal bronchial epithelial cells). However, the mRNA levels of the other three chemokines were found to be similar to the BEAS2B cell line. Moreover, plasma CCL19 levels were examined from 89 SCLC patients and compared with 10 healthy individuals, and it was shown that plasma CCL19 levels were upregulated significantly in SCLC patients compared to healthy controls. Furthermore, CCL19 concentration was found to be tremendously higher in late-stage metastasis compared to early-stage. The lymph node metastasis positively correlated with distant metastasis in all the SCLC patient cohort compared to the training cohort and validation cohort. High levels of CCL19 in plasma have negatively affected overall patient survival [32]. Knockdown of CCL19 in SCLC cell lines (H196 and H446) showed decreased proliferation, migration, invasion, and angiogenesis [32]. Interestingly, CCL19 was also shown to modulate the functioning of CD8+ T cells in SCLC (Fig. 1) [32]. The outcomes of the study provided the implications of CCL19 targeting to reduce the spread of metastatic SCLC cells, and this might lead to improve the OS of SCLC patients.

Table 1:

Chemokines regulating metastasis, heterogeneity, and immune response in SCLC.

Chemokine(s) or chemokine receptors	SCLC subtype(s)	Readout	References
CCL8	Not Defined	-CCL8, CCL19, CCL21, and CCR1, peak differentially expressed among primary lesions and metastatic lymph nodes.	[32]
CCL19		-CCL19 inhibition decreases SCLC cell proliferation, migration, invasion, and tube formation of HUVEC cells.
CCL21		-CCL19 modulates the percentage of CD8+ T cells.
CXCL8 (IL-8), CXCR1, CXCR2	Not Defined	-CXCL8 and VEGF enhance angiogenesis. -Stimulate cell proliferation of SCLC cells. -shows high specificities towards CXCR1. -anti-CXCR1 antibodies decreased cell proliferation.	[49, 56]
CXCL12, CXCR4	Comparatively, high in cell lines belong to SCLC-A/-N subtypes	-CXCR4 is a major chemokine receptor in SCLC. -Enhances metastasis. -Chemoresistance. -Stromal modulation. -Induces MAPK signaling.	[68, 69, 73, 74]
CCL2	-High in the non-NE subtype.	-Chemoattractant for monocytes. -Activates monocytes for cancer cell killing.	[86, 104]
CCL5	-High in non-NE or SCLC-I subtype	-Activation of cytotoxic T-cells. -STING agonist increases CXCL10. -MHC-I recovery in the non-NE subtype.	[104, 106]
CXCL10	-low in NE or SCLC-A/-N subtype	-Improved antigen presentation. -Induces strong anti-tumor immunity.

2.2. Tumor heterogeneity in SCLC

LC is one of the foremost causes of cancer-related deaths across the globe. One of the most significant challenges for clinicians while treating SCLC patients is relapse. The patients initially respond well to the chemotherapy, but cancer soon resurfaces. Intra-tumoral heterogeneity (ITH) may contribute to this treatment failure in SCLC patients. ITH refers to distinct cell populations within the tumor regarding their molecular and phenotypic profiles. These special subcellular populations within tumor explain the differences in the tumor growth rate, drug sensitivity, invasion, genetic variations, and prognosis. Understanding tumor heterogeneity is crucial in developing a tailored treatment for a patient. The lack of biopsy samples limits the studies of tumor heterogeneity in the SCLC [58]. Due to these challenges, SCLC was treated as a single disease for a long time.

Initially, SCLC was divided into two types: classic and variant. This classification came into existence after years of generation and classification of SCLC cell lines by Minna and Gazdar groups [59–61]. Classic SCLC cell lines which accounted for 70% of the total cell lines, showed elevated levels of neuroendocrine markers L-dopa decarboxylase, bombesin-like immunoreactivity, brain isozyme of creatine kinase, and neuron-specific enolase. The variant cell type lacked expression of the first two NE markers but tended to have MYC amplification. Another clue towards the possible molecular heterogeneity in SCLC came from a study conducted on a panel of cell lines to check their susceptibility to Seneca Valley Virus (SVV), a neuroendocrine cancer-selective oncolytic picornavirus [62]. It was observed that ASCL1 and NEUROD1 were among the most differentially expressed genes in the permissive and nonpermissive cell lines. Recent advancements in genomics, proteomics, and transcriptomics analysis, have paved the way for a new molecular classification of SCLC subpopulations. The classification is based on the differential expression of transcription regulators: ASCL1, NEUROD1, POU2F3, and YAP1. A recent study published by Rudin et al. proposed a working nomenclature for the SCLC molecular subtypes dividing them into four categories: SCLC-A (ASCL1), SCLC-N (NEUROD1), SCLC-P (POU2F3), and SCLC-Y(YAP1) [11]. Among them, SCLC-A comprises approximately 70% of human SCLC.

It has been observed in multiple RB1 and TP53 mutant mouse models that the SCLC-A originates from the pulmonary neuroendocrine cells (PNECs) and shows higher expression of NE markers such as chromogranin A and synaptophysin [13, 63]. It has been established that loss of tumor suppressor genes RB1 and TP53 results in tumor progression of SCLC. However, the amplification of MYC family members is not universal in the SCLC subtype. SCLC-A exhibits an enriched expression of MYCL and MYCN and lower expression of MYC, unlike other SCLC subtypes. This variability stems from that MYC and MYCL are functionally distinct, with different epigenetic profiles and positions in the gene loci [64]. It is also recognized that ASCL-1 and Notch were present in mutual antagonism. SCLC-A subtype shows a higher expression of Delta-like ligand 3 (DLL3), a Notch pathway inhibitor downstream target of ASCL1 [65]. It is well established that activation of notch signaling pathways inhibits the neuroendocrine differentiation of precursor cells [66].

Of late, Ireland et al. conducted a study where they observed that MYC shifts NE subtype to non-NE through Notch-signaling [6]. Notch mutant SCLC tumors were locked in a NE-state exhibiting the genomic and phenotypic characteristics of the SCLC-A subtype. However, in the Notch wild-type tumor, there was a temporal shift from the ASCL1⁺ state to the YAP1⁺ state mediated by MYC. Similar results were obtained from the gene set enrichment analysis (GSEA) of notch signaling, which revealed MYC induction of NOTCH2 and HES1 protein levels [1]. HES1 is also overexpressed in ASCL1+ non-NE SCLC cells which serves as an indication of the extensive intra-tumoral heterogeneity in SCLC tumors. MYC also regulates the TIME in lung adenocarcinomas. Higher expression of MYC induces certain chemokines and cytokines. For example, CCL9 and IL-23 are induced by MYC in KRAS/MYC mutant lung adenoma mouse models [67]. CCL9 is a ligand of CCR1, which is present on T cells, monocytes, and MDSCs. The CCR1-CCL9 axis is involved in tumor progression and metastasis in lung adenocarcinomas [67].

There is a lack of information on the subtype-specific secretion/expression of chemokines in SCLC as no in-depth or comprehensive studies are available describing the subtype plasticity or heterogeneity in the context of chemokines or chemokine receptors. However, few studies established the mechanistic role of the CXCL12/CXCR4 axis in SCLC metastasis, chemoresistance, and stromal modulation (Table 1) [68–73]. These studies were conducted before the subtype-specific classification of SCLC (SCLC-A/N/Y/I/P); therefore, if we revisit the outcomes of these studies in correlation with the recent classification of SCLC cell lines, it can be pragmatic that the expression of CXCR4 or CXCL12 is higher in NE-subtype as compared to non-NE subtype cell lines. The high CXCR4 expression was reported in NCI-H69, NCI-H82, NCI-H146, and SBC5 cells (NE or subtype-A/N cell lines) compared to SBC3 cells (belonging to non-NE or subtype-Y/P) [68, 69, 74]. As a result, these studies provided preliminary clues for the involvement of chemokines in regulating the plasticity or heterogeneity of SCLC. However, the detailed profiling of chemokines or chemokine receptors is a highly desired and crucial area of future investigations that will elucidate how chemokines impact or regulate SCLC heterogeneity and illuminate the novel therapeutic vulnerabilities (Fig. 2).

Figure 2: Chemokines in SCLC heterogeneity and therapeutic immune responses.

SCLC has a high heterogeneity and is majorly divided into five subtypes SCLC-A/-N/-Y/-I/-P. Chemokines modulate the therapeutic response in various subtypes of SCLC. Targeting of SCLC using PARP/CHK1, STING, WEE1, and LSD1 inhibitors induce the expression of CCL5, CXCL10, and PD-L1 in SCLC, enhancing the chemo-immunotherapeutic response in SCLC (extreme left panel). In terms of the immune response or the status of active immune cells, SCLC-A/-N subtypes (neuroendocrine) are considered immune cold tumors, whereas SCLC-Y/-I/-P subtypes (non-neuroendocrine) are considered as immune hot tumors. The chemokine status in immune cold SCLC tumors remains elusive (upper right-hand panel). Immune hot SCLC tumors produce CCL5 and CXCL10 that help to recruit NK cells and T-cells (cytotoxic) and enhance the immunotherapeutic response of anti-PD-L1 or anti-PD1 immunotherapies (lower right-hand panel).

2.3. Chemokines in SCLC metastasis

SCLC is a very aggressive NE tumor, characterized by fast tumor growth, therapeutic resistance, high genomic abnormalities, and early metastasis. More than 70% of the patients that are diagnosed with SCLC show macro-metastases in the brain, bone, liver, and lymph nodes. Spontaneous mouse models with TP53 and RB1 deletions (RP models) show metastases to the liver, lymph nodes and other organs, including the spleen and kidney [75, 76]. The members of the MYC family are also amplified in human SCLC. Incorporating constitutively active MYC gene in the RP along with RB1 and TP53 deletion (RPM model) shows rapid SCLC tumor growth that metastasizes within a few weeks after initiation [7, 77]. These genetically engineered mouse (GEM) models, patient-derived xenografts (PDX), and circulatory cell-derived xenografts (CDX) models have led to a better understanding of the cellular and molecular mechanism behind SCLC metastases; however, the possible role of multiple chemokines in SCLC metastasis still needs to be explored comprehensively.

Few studies have identified the role of chemokines in the metastasis of SCLC cells, and the most studied pair is CXCR4/CXL12 axis [68, 70, 73]. Apart from CXCL12, CCL19 has also promoted the migration of SCLC cells. Recently, Liu et al. observed a significant increase in the expression of CCL19 in the lymph node metastatic lesions compared to the normal tissues [32]. It was observed that CCL19 was associated with proliferation, invasion, migration, poor patient survival, and HUVEC tube formation (Table 1). An increase in the VEGF-C and VEGF-D in response to hypoxia-induced overexpression of CCR7 in cancer cells leads to lymph angiogenesis [78]. As a result, cancer cells enter the lymphatic vessels and migrate to the lymph node. Since lymph nodes show higher expression of CCL19, the CCR7-expressing cancer cells (including SCLC) get trapped and metastasize. CCL19 also recruits Treg cells to the tumor niche, which inhibits the anti-tumor response of the immune system [32, 79]. Given the importance of CXCR4/CXCL12 and CCR7/CCL19 in promoting and establishing SCLC metastasis, it will be imperative to investigate the impact of chemokines in SCLC metastasis, and it would be an important area of future SCLC studies (Fig. 1).

3. Chemokines and targeted therapies of SCLC

To this day, chemotherapy has been the first-line of therapy for SCLC patients, with a median survival time between 7–10 months [80]. Combination chemotherapy using etoposide-platinum has become the preferred choice for first-line treatment, and relapse occurs quickly with reduced sensitivity to chemotherapy. However, recently much interest has emerged in developing a combinatorial approach to chemotherapy and immunotherapy, and many of the initial trials have already been completed with some positive (iMpower 133 and CASPIAN) and negative (CheckMate 331 and 451) results [81]. Unfortunately, studies targeting chemokines and their receptors in SCLC has been scarce, and only a few small molecules have been tested [73, 82]. Various antibodies targeting chemokine receptors have not been investigated in SCLC, whereas many of those antibodies have already been studied for other cancer types [45, 73, 82–85]. Here we put together the potential of some of the chemokines and their receptors, which could be the putative targets for therapy development in SCLC.

The chemokine CCL2 is drastically reduced in SCLC, which serves as the chemoattractant signaling molecule for monocytes and also activates them to kill malignant cells [86]. Mass array methylation study demonstrated that the reduced expression of CCL2 is regulated by H3K27me3/tri-methylation in the enhancer region, which was mediated by EZH2, a part of the PRC2 complex, and DNA methylation of the promoter regions through DNA methylating enzyme DNMT1 [64, 87]. Two small molecule inhibitors, including decitabine and EPZ011989 have shown effectiveness in reverting these conditions [87]. Using an SCLC ectopic engraft model, Zheng et al., have shown that combination treatment using the above mentioned two drugs has potentially disrupted DNMT1/EZH2 mediated repression of CCL2 and this led to abrogate the inhibition that was imposed on macrophage infiltration [87]. It is interesting to mention that a high level of macrophages has been observed in SCLC patients with better overall survival (OS) [88]. Consequently, tumor growth was suppressed, and the opposite scenario was observed with the depletion of macrophages and CCL2 downregulation. So, targeting CCL2 can be one novel treatment strategy that can be investigated for clinical use, and a good clinical outcome might come from it.

It has been reported that the CXCR4 receptor, which uses chemokine CXCL12 as its ligand, is overexpressed in SCLC patient samples and serves as the major chemokine receptor in primary SCLC cells [69, 73]. Moreover, CXCL12 induces endocytosis of CXCR4 in SCLC cells and subsequent activation of MAPK signaling via P-p44/42 mitogen-activated protein kinase. It further promoted the invasion of malignant cells in ECM and the strong adhesion of SCLC cells to marrow stromal cells. This adhesion was found to be impaired by T140, a CXCR4 antagonist. Therefore, CXCR4 targeting can be a potential site for intervention. Taromi and coworkers have studied the inhibition of the CXCR4-CXCL12 axis in their orthotopic xenograft mouse model that closely resembles extensive SCLC in clinical settings and have shown that AMD3100 treatment (a specific inhibitor of CXCR4) decreases the proliferation of the primary tumors in those mice models by 61% (P<0.05) and reduced the occurrence of metastasis by 43% [82]. On the other hand, cisplatin and etoposide combination therapy decreased the growth of primary SCLC tumors by 71% but could not prevent metastasis. When chemotherapy and CXCR4 inhibitor were combined for therapy, it reduced tumor growth to a margin similar to combination chemotherapy and prevented the metastasis like AMD3100 monotherapy. This mouse model study showed that combining chemotherapy with a CXCR4 antagonist could reduce SCLC metastasis. In conclusion, combination therapy has enormous potential in clinical application to enhance overall patient survival and quality of life in SCLC patients.

CXCR4 pathway has been targeted with different antibodies and has generated impressive outcomes against multiple types of cancer. For instance, PF-06747143 (novel humanized anti-CXCR4 IgG1 antibody was used in chronic lymphocytic leukemia (CLL) targeting the CXCR4 pathway and it promoted the death of the primary CLL-B cells (patient-derived), with or without stromal cells. Cell death after PF-06747143 treatment had signs of programmed cell death. It was also combined with standard drugs (rituximab, fludarabine, ibrutinib, and bendamustine) and found to have significant outcomes. They also observed that this drug is effective in reducing tumor burden and improving OS as a stand-alone therapy or in combination with bendamustine in the xenograft mouse model of CLL [84]. Ghobrial et al. have tested another anti-CXCR4 antibody named Ulocuplumab in combination with other standard drugs in relapsed multiple myeloma patients with an average clinical benefit rate of 72.4% [83]. Some of those patients had previously received immunomodulatory agents. Another anti-CXCR4 antibody called MDX1338 was used in sarcoma cells along with natural killer cells (activated and expanded) that led to inhibition of metastasis in mouse models [85]. LY2624587, another humanized anti-CXCR4 agent, was found to be inducing apoptosis in hematologic malignancies in both in vitro and xenograft models. However, none of these antibodies have been tested on SCLC cell lines or any mouse models [89]. With so many anti-CXCR4 antibodies already being successfully studied in different malignancies, it is of paramount importance to test them on SCLC cell lines and patients as these antibodies are already in clinical trials and may help to reduce the burden of SCLC.

4. Chemokines in immune response and immunotherapy

Chemokine not only participates in the growth and metastasis of various cancers, but they have a multifaceted role in tumor immunity [29, 31, 90, 91]. Given a dynamic and diverse expression of chemokine receptors and ligands by immune and stromal cells, chemokines orchestrate key features of immune cell biology during tumorigenesis, such as immune cell recruitment, phenotype switching, and activation [29, 31, 91, 92]. Interestingly, it was demonstrated that high tumor mutational burden (TMB) positively correlates with neoantigen production and concordantly increases the favorable immune response to immune checkpoint inhibitors blockade (ICB) such as anti-PD1/PD-L1 in different cancers, including NSCLC [93–97]. The primary cause of SCLC is cigarette smoking or tobacco carcinogens that adducted DNA and contribute high TMB to SCLC due to the accumulation of C>A transversions, and owing to the high mutational burden of SCLC, it was anticipated that this might correlate with the production of the high amount of neoantigens and enhanced immune response [1, 98]. However, the positive correlation between high TMB and neoantigens production does not portray in SCLC [98, 99]. Most of the SCLC subtypes showed restricted infiltration of immune cells, and it remained largely unclear whether this immune cold behavior of SCLC was credited to defects in antigen presentation or the absence of tumor antigens [13, 15]. Therefore, the unpredicted immune cold behavior of SCLC remains a dynamic area of investigation (Fig. 2).

An interesting study unveiled the immune evasive behavior of SCLC and showed that SCLC cell lines secreted TGF beta 1 that selectively decreased the production of cytokines or chemokines from immune cells; however, the detailed investigation in SCLC patients remains to be investigated [100]. Another exciting study demonstrated that serum cytokine levels work as a predictive biomarker for ipilimumab benefits and OS of SCLC patients treated with immuno-chemotherapy [101]. Higher level of IL-1β, IL-2, IL-6, IL10, interferon-gamma, TNF-alpha, and GM-CSF has been detected in the serum samples of SCLC patients treated with chemotherapy + ipilimumab compared to patients treated with chemotherapy alone and found differentially correlated with overall survival of patients [101]. This study evaluated serum cytokines as a prognostic biomarker, and opened a window to further extend these outcomes for the analysis of chemokines in SCLC patients under different chemoimmunotherapeutic strategies to uncover the immune-suppressive behavior of SCLC. Indeed, few studies showed that, as compared to other tumors, SCLC tumors display low MHC class 1 (MHC-I) antigen (human leukocyte antigens and β−2-microglobulin) [102–104].

Interestingly, high chemokines secretion has been shown in drug-resistant (EZH2 inhibitor-resistant) SCLC cell lines (mimics the non-NE SCLC phenotype), and these cells also show increased expression of MHC-I compared to their parental phenotype (NE-phenotype) [104]. MHC-I^High SCLC cells secreted high VEGF, GM-CSF, CCL5, CCL2, and CXCL10. These resistant SCLC cell lines show sensitivity to stimulator of interferon genes (STING) agonist with enhanced production of CXCL10 and enforced the antigen presentation or MHC-I recovery in non-NE SCLC phenotype that further activates intrinsic, innate immune response/signaling (Table 1) [104]. Supporting these observations, a fascinating recent study provided the immune landscape of SCLC patients (subtype-specific) through bulk transcriptional profiling [13]. The expression of MHC-I, PD-L1, and T cell attractant chemokines (CCL5, CXCL10) were suppressed in SCLC-A/N (NE tumors), whereas SCLC-I (inflamed, a non-NE phenotype) experienced high CCL5 and CXCL10 expression with enhanced response to immuno-chemotherapy (Fig. 2, Table 1) [13].

Chemokines also modulate the therapy response of drugs targeting DNA damage response (DDR) pathways in the SCLC [105]. It has been shown that the targeting of PARP or checkpoint kinase 1 (CHK1) increased the cell surface expression of PD-L1, and the inhibition of PARP or CHK1 augmented the infiltration of cytotoxic T-cells in immunocompetent in vivo models of SCLC [105]. The mechanistic segmentation of DDR inhibition mediated immune response established that it activates STING/TBK1/IRF3 immune pathway with the increased levels of CCL5 and CXCL10 chemokines, which accomplished the activation of cytotoxic T-cells and enhanced the anti-tumor immune response in SCLC [105]. Taniguchi et al. recently investigated that inhibition of WEE1-activated STING and STAT1 pathways, and WEE1 inhibitors in combination with ICB modulate the innate and adaptive anti-immune response, increased the expression of type I interferons (IFN-α/β), and CCL5 and CXCL10 chemokines in SCLC [106]. The CCL5 and CXCL10 chemokines induce strong anti-tumor immunity in SCLC cells (in vitro and in vivo murine models). One of the interesting observations of this study is that WEE1 inhibitors increased the cell surface expression of MHC-I in human and murine SCLC cell lines; however, further investigations are needed that can explain whether MHC-I expression is coupled with or regulated by the increased expression of CCL5 or CXCL10 (Fig. 2). Interestingly, targeting cyclin-dependent kinase 7 (CDK7) via YKL-5–124 resulted in activating immune responses in SCLC via releasing chemokine CXCL10 and has further shown encouraging tumor responses with immunotherapy in SCLC GEM models [107]. Recently, it has been documented that high metalloproteinase 9 (MMP9) activity in immune cells is associated with chemokine binding as compared to low MMP9 expressing SCLC tumors [108]. The exciting outcomes of discussed studies suggest that chemokines may serve as an additional biomarker to stratify SCLC patients to immunotherapies and predict immune cell infiltration in SCLC tumors. However, a small subset of SCLC-A showed high HLA and NK cells and T cell scores, suggesting that there is an urgent need for the identification of additional biomarkers that can withstand chemokines and other biomarkers to stratify patients for immunotherapy alone or immuno-chemotherapy [13, 109].

5. Concluding remarks and future directions

Progressive advances in SCLC biology and the outcomes of high throughput studies such as single-cell sequencing, and bulk transcriptional studies reevaluate SCLC classification that metamorphosed the classical view of homogenous SCLC into highly dynamic and heterogenous disease with multiple molecular subtypes (SCLC-A/N/Y/I/P) [14]. However, due to under-treasured inter-/intra-tumoral heterogeneities, high metastasis, recurrence, chemoresistance, and low immune infiltration, SCLC remained a notorious cancer type with various proven challenges. The mechanistic dissection of drug resistance uncovered that subtype plasticity is the primary reason for chemo-/immuno-therapy resistance [13, 14, 104]. The other major contributing factors were epigenetic rearrangements/modulations and decreased expression of immune gene signatures (such as MHC-I, PD-L1, HLA, and β−2-M), further limiting the chemotherapeutic or immune response and the production of immunogenic antigens [104].

In addition to the major transcription factors or subtype regulators (ASCL1, NEUROD1, YAP1, POU2F3, and PLCG2), chemokines and chemokine receptors play a multilayered role in various aspects of SCLC, including metastasis, drug resistance, and immunogenicity. Given the role of chemokines in metastasis and cancer cell to immune cell or cell-cell interactions, chemokines meticulously regulate anti- and pro-SCLC immune response and therapy resistance. Chemokines deliver an anti-tumor immune response to TME and regulate the recruitment of Treg (pro-tumorigenic), a primary immunosuppressive cell to the TME of various cancers, including SCLC. The secretion or expression of selective chemokines can be concomitant with therapy responses in SCLC, such as CCL5 and CXCL10 and can be utilized as a biomarker for predicting the ICB+ chemotherapy response and stratification of SCLC patients for chemo-immunotherapies. However, the dual participation of some of the chemokines (pro- and anti-tumor) makes it an interesting landscape that needs to be investigated in the context of overall tumor immune response. Exciting new studies demonstrated that the secretion or expression of some of the chemokines or cytokines (such as VEGF, GM-CSF, CCL2, CCL5, and CXCL10) were subtype-specific and play a key role in modulating chemo-immune response and combinatorial targeted/ICB therapy response [13, 104–106]. However, the mechanism regulating the cascade of these chemokines in SCLC remains elusive. It provides a potential area of future research with the renovated hope that the dissection of these mechanisms can be implicated in improving the chemo-immunotherapeutic responses. The overall panorama of chemokines in SCLC is understudied. It needs detailed/multicentered studies that can provide the various aspects of chemokines in SCLC biology, such as heterogeneity, subtype plasticity, metastasis, and chemoresistance that can be traveled to improve the therapeutic response as well as for the identification and development of novel therapies to conquer this deadly disease.

Abbreviations

ASCL1

Acheate-scute homologue 1

CDX

circulating cell line derived xenografts

CHK1

checkpoint kinase 1

CLL

chronic lymphocytic leukemia

DDR

DNA damage response

GEM

genetically engineered mouse

HLA

Human Leukocyte Antigens

IL

interleukin

ILCs

innate lymphoid cells

ICB

immune checkpoint blockade

LC

lung cancer

MYC

Myc proto-oncogene protein

MDSCs

myeloid-derived suppressor cells

MHC

major histocompatibility complex

NE

neuroendocrine

NeuroD1

neurogenic differentiation factor 1

non-NE

non-neuroendocrine

NFIB

nuclear factor IB

ORR

overall response rate

OS

overall survival

PDX

patient-derived xenografts

pDCs

plasmacytoid dendritic cells

PRC

polycomb repressive complex

POU2F3

POU class 2 homeobox 3

PFS

progression free survival

PNECs

pulmonary neuroendocrine cells

SCLC-A

high ASCL1

SCLC-N

high NEUROD1

SCLC-P

high POU2F3

SCLC-Y

high YAP-1

SCLC

Small cell lung cancer

STING

stimulator of interferon genes

TIME

tumor immune microenvironment

TME

tumor microenvironment

TMB

tumor mutational burden

TS

tumor suppressor

VEGF

vascular endothelial growth factors

YAP1

yes associated protein 1