Mechanisms of delayed Indocyanine Green fluorescence and applications to clinical disease processes

Abstract

Background:

Delayed indocyanine green (ICG) fluorescence imaging is under investigation in various clinical disease processes. Understanding the mechanisms of ICG accumulation and retention is essential to correctly interpret and analyze imaging data. The purpose of this scoping review was to synthesize what is known about the mechanism of ICG retention at the cellular level to better understand the clinical nuances of delayed ICG imaging and identify critical gaps in our knowledge to guide future studies.

Methods:

We performed a scoping review of 7087 citations after performing database searches of PubMed, Scopus, The Cochrane Library and the Web of Science Core Collection electronic databases. Studies were eligible for inclusion if they were peer-reviewed original research discussing the mechanism of ICG retention in the results section in disease processes involving inflammation and/or necrosis, including cancer, and were available in English. Data was extracted using Covidence software.

Results:

Eighty-nine studies were included in the final analysis. Several features of ICG retention were identified.

Conclusions:

We identified several mechanistic features involved in ICG accumulation in diseased tissue that overall demonstrate distinct mechanisms of ICG retention in tumors, non-tumor inflammation and necrosis. Our study also reveals new insight on how inflammatory infiltrate influences ICG fluorescence imaging. These findings are noteworthy because they add to our understanding of how fluorescence-guided surgery may be optimized based on the pathology of interest via specific ICG dosing and timing of image acquisition.

INTRODUCTION

In vivo fluorescence imaging is an ongoing area of interest for surgeons because it has a high signal-to-background ratio, allows for overlay of white-light and fluorescence images, may be used with operating room lighting, utilizes small concentrations of highly sensitive, targeted molecular dyes, has quantitative capabilities, and is easy to use relative to other imaging technologies.^1,2 Indocyanine green (ICG) fluorescence imaging, specifically, has emerged as a leading real-time surgical technology with applications in many surgical subspecialties. This is especially demonstrated by a dramatic increase in publications over the past decade.² ICG is an amphiphilic molecule first approved for clinic use in 1956 by the Food and Drug Administration.^1,3 The absorption and emission peaks for ICG fall within the near-infrared (NIR) spectrum (750- ~1400 nm).^4,5 The NIR fluorescence light works in the tissue optical window, penetrating several millimeters, allowing visualization of tissue below the surface but not deep structures.^1,6

Delayed imaging of ICG was first described in 2009 after residual ICG was incidentally identified in hepatic tumors up to 14 days after ICG administration for routine liver function testing.⁷ The use of delayed imaging after ICG administration, now called “second window ICG” (SWIG), is an emerging surgical navigation imaging technique in which ICG directly identifies pathological tissue and delineates surgical margins in real time. It allows for static imaging without the need for additional ICG dosing, as opposed to ICG angiography (ICGA), which is time-sensitive, dynamic imaging of perfusion, requiring repeated dosing to obtain subsequent images of the anatomy of interest (Figure 1). SWIG is under clinical investigation in disease processes containing inflammation and necrosis (NCT04084067, NCT04723810, NCT02280954, NCT01335893) despite a lack of a clear understanding of the mechanism.

Figure 1.

ICG remains intravascular during ICG angiography (ICGA) imaging of perfusion. Enhanced permeability of damaged blood vessels allows for delivery of ICG into the perivascular tissue where it is retained through cellular and tissue processes, making second window indocyanine green (SWIG) imaging feasible. Created with Biorender.com.

After intravenous injection, ICG most commonly complexes with lipoprotein (LP) and is delivered to inflammatory and tumor tissue non-specifically through extravasation from permeable blood vessels, a result of excess angiogenesis, dysfunctional endothelial cells and wide fenestrations (Figure 1).^8–18 It is then retained in the surrounding tissue due to disrupted cellular and tissue drainage pathways.^19,20 Many studies rely on this enhanced permeability and retention (EPR) effect without providing evidence or further insight into the mechanism of ICG retention despite cancer, inflammation and necrosis having distinct pathophysiology.

Given that delayed ICG imaging is becoming more commonly used for many surgical applications to image cancer, necrosis and inflammation, understanding the molecular mechanism of ICG accumulation and retention in pathological tissue is essential to correctly interpret and analyze fluorescence imaging data. The objectives of this study were to 1) review original research on the mechanism of ICG retention in cells, tissues, animals and humans with disease processes involving inflammation and/or necrosis, 2) synthesize a scoping review describing the mechanism of ICG retention within pathological tissue, and 3) report on whether cancer and other clinical disease processes involving inflammation and necrosis have common or disparate mechanisms of ICG retention.

A scoping review was the study design that best aligned with our objectives, as scoping reviews are designed to provide an overview of a heterogeneous body of literature on a broad topic, identify and map concepts, clarify knowledge gaps, and evaluate areas of future research. The goal of this body of work was not to comment on current practice, but rather to synthesize current research on ICG retention in pathological tissue and identify conceptual boundaries as a focus for future scientific investigation.

METHODS

Protocol

The protocol was developed using the scoping review methodological framework proposed by Arksey and O’Malley (2005).²¹ The draft protocol was revised upon receiving feedback from the research team, including a university librarian and the authors.

Eligibility Criteria

Studies were eligible for inclusion if they were peer-reviewed original research on the mechanism of ICG retention in cells, tissues and/or organisms (animal or human) with disease processes involving inflammation and/or necrosis, including cancer. Exclusion criteria included: 1) non-original research including commentaries, editorials, reviews and meeting proceedings, 2) published abstracts without peer-reviewed manuscripts, or clinical trial registrations, 3) ICG was utilized as a “tattoo” (i.e., ICG was administered as a localized injection through the skin or endoscopy/colonoscopy) in which there was no focus on its mechanism of retention, 4) ICG was studied in healthy tissue, 5) imaging was performed less than 15 minutes after ICG administration, 6) absent discussion on ICG’s mechanism of retention in the results section, 7) ICG was bound to another molecule (non-physiologic), and 8) inability to obtain the full text. Articles published in languages other than English were excluded because of limited resources for translation.

Database sources and search strategy

Comprehensive literature searches were conducted by a university librarian in consultation with the research team. PubMed, Scopus, The Cochrane Library and the Web of Science Core Collection electronic databases were searched on April 6, 2022 and December 23, 2022, yielding 7087 studies across all databases after removing 134 duplicate studies. Covidence (Melbourne, Australia) was used for citation management.

Study selection

A two-stage screening process was used to assess the relevance of studies identified in the search. For the first screening level, only the title and abstract of citations were reviewed to procure articles that met eligibility criteria. The title and abstract of each citation (n = 7087) were independently screened by two reviewers. The reviewers were not masked to author or journal name. A third reviewer (senior author of study) resolved all conflicts. All citations included after title and abstract screening were procured for full text screening. Two reviewers independently screened each manuscript (n = 803). Reviewers met throughout the screening process with the senior author to resolve conflicts and discuss any uncertainties related to study selection.

Data extraction

Eighty-nine studies were selected for inclusion after the two-stage screening process. A data extraction form with study characteristics including title, publication year, authors, type of study, and model, ICG imaging details including dose and imaging timing, overall outcomes, and mechanistic details was created within Covidence and used to extract data from each included study. The data were exported and imported into Microsoft Excel (Microsoft Corporation, Redmond, WA) (Supplemental Table 1).

RESULTS

General characteristics of included studies

Eighty-nine studies were included in the analysis. The PRISMA diagram of the flow of articles through identification to final inclusion is represented in Figure 2. Table 1 describes the general characteristics of the 89 studies included for review. All included studies were published between 1996 and 2022, and varied widely in terms of disease process studied and study design. Of the 89 studies, ICG retention was discussed in 75 studies involving tumors, 23 studies on inflammatory pathologies, and 11 in necrotic tissue. 47 studies were basic science, 6 were case reports, 6 were case series, 19 were non-randomized experimental human studies, and 11 were translational studies, including multiple experimental models spanning from single cell cultures to human subjects. Several main themes describing the mechanisms of ICG retention were identified and will be described below (Table 2 and Supplemental Table 2).

Figure 2.

PRISMA diagram of the flow of articles through identification to final inclusion.

Table 1.

Study characteristics of included manuscripts.

Characteristic		Number (n=89)	Percentage (%)
Publication year	<2000	1	1
	2000–2009	4	4
	2010–2019	57	64
	2020-December 2022	28	31
Country of publication	United States	29	32.6
	Japan	22	24.7
	China	14	15.7
	Canada	4	4.5
	Korea	4	4.5
	The Netherlands	4	4.5
	The United Kingdom	3	3.4
	Taiwan	3	3.4
	Austria	2	2.2
	France	2	2.2
	Germany	2	2.2
Study design	Basic science	47	52.8
	Case report	6	6.7
	Case series	6	6.7
	Non-randomized experimental study	19	21.3
	Translational	11	12.4
Study model	Hepatocyte-derived tumor	18	20.2
	Non-hepatocyte tumor	56	66.3
	Inflammation	23	25.8
	Necrosis	11	12.4
Timing of delayed imaging after ICG application (cells) - incidence	15–59 min	8	9.0
	1 hour	8	9.0
	1.5–11 hours	10	11.2
	12–23 hours	4	4.5
	24 hours	14	15.7
	48+ hours	3	3.4
Timing of delayed imaging after ICG injection (animal/humans) - incidence	15–59 min	17	19.1
	1 hour	12	13.5
	1.5–11 hours	24	27
	12–23 hours	14	15.7
	24 hours	50	56.2
	48+ hours	30	33.7

Table 2.

Mechanisms of delayed ICG retention in healthy and pathologic tissue.

	ICG Mechanism	Disease	ICG Dose	Method of ICG Administration	Timing of ICG Imaging After ICG Administration	References
Cancer	Cellular uptake of ICG is dependent on tight junction disruption	Colon cancer	25 μM (cell culture); 7.5 mg/kg (animal)	Incubation for 15 min (cell culture); topical & IV (animal)	15 min (cell culture); 0–28 h (animal)	16,18
	ICG binds the cytoplasmic membrane	Squamous cell carcinoma, colon cancer, hepatocellular carcinoma	1 – 100 μM (cell culture); 10 μmol/kg or 7.5 mg/kg (animal)	Incubation for 15 min - 24 h (cell culture); IV (animal)	15 min (cell culture); 0–28 h (animal)	16,18,23,67
	ICG binds to necrotic cells, likely through binding the damaged phospholipid membrane	Breast cancer, sarcoma	0.5–3 mg/kg (animal)	IV	24 h	24,25
	ICG is internalized based on a concentration gradient (diffusion) after interacting with the cellular membrane	Squamous cell carcinoma, breast cancer, metastatic cancers to the liver, hepatocellular carcinoma, glioma, sarcoma, pancreatic cancers, colon carcinoma, melanoma, cervical cancer	1 – 100 μM (cell culture); 1–20 mg/kg (animal); 0.25–5 mg/kg (human)	Incubation for 1, 4 or 24 h (cell culture); IV (animal & human)	0.5–24 h	9,11,12,16,18,27,51,54,55,67–69
	ICG is internalized via endocytosis and is transported via the membrane trafficking system	Head and neck squamous cell carcinoma, cervical cancer, lung carcinoma, colon carcinoma	100–1600 ng/mL (cell culture); 7.5 mg/kg (animal)	Incubation for 10 min - 24 h (cell culture); IV (animal)	10 min - 28 h	16,18,31,53,70–72
	ICG is metabolized in healthy tissue faster than pathologic tissue resulting in retention in tumors	Breast cancer, pituitary adenoma, cervical cancer, hepatocellular carcinoma	50 uL of 50 mM (animal); 0.5–5 mg/kg (human)	Intratumoral or subcutaneous injection (animal); IV (human)	3 min - 48 h; 2–14 d (hepatocellular carcinoma specifically)	10,46,73,74
	ICG is retained due to impaired biliary excretion	Hepatocellular carcinoma, melanoma and colorectal liver metastases	0.05–10 mg/kg (animal & human)	IV	3–96 h (animal); 1d - 8 weeks (human)	7,29,38,42–45,47,52,62,63,65
	NTCP, OATP8 and OATP1B3 transporters internalize ICG	Hepatocellular carcinoma, breast cancer, sarcoma, colorectal adenocarcinoma	0.4–300 μg/ml (cell culture); 200 μg or 8 mg/kg (animal); 0.5 mg/kg (human)	Incubation (cell culture); IV (animal & human)	1–4 h (cell culture); 1h -14 d (animal & human)	29,57–59
	ICG binds cytoplasmic proteins	Glioma	0.4 mg/kg (animal)	IV	40 min	32
	Canalicular transport of ICG is mediated through the MDR3 transporter (in hepatocytes)	Hepatocellular carcinoma	Not reported (human)	IV	0–4 weeks	56
Inflammation	ICG is internalized via phagocytosis	Atherosclerosis, colorectal liver metastases, ocular inflammation	0.25–10 mg/kg (animal); 0.5 mg/kg (human)	IV	20 min - 72 h	14,22,34–37,40,41
	ICG accumulation is not solely due to increased blood flow at the infectious/inflammatory sites	Streptococcus pyogenes inflammation	0.25 mg/mL (animal)	IV	0–24 h	75
	ICG is retained in hepatocytes due to sepsis-mediated canalicular excretion dysfunction	Polymicrobial peritoneal infection	14 pmol/g (animal)	IV	30 – 300 min	60
	ICG is retained in tissue with disrupted vasculature	Sarcoma inflammation, colitis, skin lesions	3–7.5 mg/kg (animal); 0.25 mg/kg (human)	IV or intradermal (animal); IV (human)	1–24 h	14,15,27,76
	Microtubule networks are involved in ICG cytoplasmic transport	Ischemic-reperfusion	0.5 mg/kg (animal)	IV	0 – 24 h	61
	ICG is metabolized through lymphatics	Ocular inflammation	1 mg (animal)	IV, intraperitoneal	72 h	35
Necrosis	ICG has necrosis avidity through binding disrupted cellular membranes	Glioma, burn, sarcoma	0.5–3 mg/kg (animal); 2.5 mg/kg (human)	IV	5 min - 9 d (animal); 24 h (human)	9,24,25
	ICG undergoes cellular uptake due to loss of cellular barrier function	Head and neck squamous cell carcinoma, dermal necrosis, burn	100–500 ng/mL (cell culture); 1–5 mg/kg (human)	Incubation (cell culture); intradermal or IV (human)	10 min - 48 h	26,30,31

Binding to viable cell membranes is via free ICG. All other mechanisms involve ICG bound to protein (ICG-LP complex). Abbreviations: ICG = indocyanine green; IV = intravenous; min = minutes; h = hour; d = day.

ICG diffusion through tissue is influenced by disrupted tight junctions

Enhanced permeability of blood vessels results in extravasation of ICG into inflamed and cancerous tissue through dysfunctional endothelium, but little is known about how inflammation specifically influences ICG accumulation. Out of the 89 included studies, 23 studied the relationship between ICG retention and local tissue inflammation. Tissue with ICG accumulation has increased prostaglandin E2, COX-2, iNOS, and HO-1, suggesting that inflammatory mediators positively influence ICG delivery and thus retention.¹⁵ In atherosclerosis studies of ICG retention, endothelial cells located at the luminal surface of the intimal layer did not retain ICG signal over time, confirming ICG extravasation from blood vessels into inflamed tissue.²² Additionally, ICG was limited to the outer layers of thicker atheromas,¹⁴ suggesting diffusion limitations of ICG tissue penetration.

More specifically, cellular tight junction integrity has been shown to be inversely correlated with ICG uptake.^15,16,18 Tumor cells with intact tight junctions and non-disrupted occludin expression did not exhibit ICG fluorescence signal,^15,16,18 suggesting that disrupted tight junctions allow ICG retention through increased tissue diffusion capacity by which ICG leaks into interstitial space. This is consistent with what is known about the enhanced permeability and retention effect in that poor capillary architecture with abnormal basement membranes and fissures between endothelial cells allows ICG to leak out of blood vessels and into peripheral tissue through diffusion.¹¹

ICG is retained in necrotic tissue due to cellular membrane dysfunction and binding (necrosis avidity)

Two studies utilizing single cell and animal models explored ICG retention in necrosis due to tumors, reperfusion injury or burns, and further supported the hypothesis that ICG accumulates in necrotic tissue. ^23,24 In an in vivo animal model, free ICG had poor specificity for binding to tumor cells, and the preferential ICG uptake by tumor cells was regulated by the non-specific cell membrane-binding ability of ICG.²³ Likewise, free ICG in PBS bound to both viable and necrotic cells in vitro.²⁴ However, ICG complexed with lipoprotein (ICG-LP) had increased affinity for necrotic cells compared with free ICG, and the affinity increased in the presence of supplemented phosphatidylcholine, the most common exposed phospholipid tail in cell membranes damaged from necrosis.²⁴ Furthermore, the ICG-LP complex did not bind to cell membranes of viable cells, and was hypothesized to bind to the exposed hydrophobic phospholipid tails in cell membranes damaged from necrosis, suggesting a completely different mechanism of ICG retention based on necrosis affinity (Figure 3A).²⁴ This also implies that the bound status of ICG could contribute to whether viable cells demonstrate ICG retention, which is particularly relevant for in vivo studies as ICG almost completely binds to proteins within the blood stream such as LP and albumin.

Figure 3.

Mechanisms of ICG retention in (A) necrosis, (B) inflammation, (C) cancer and (D) hepatobiliary disease. Created with Biorender.com.

Eleven studies of various study designs examined ICG retention in necrotic tissue. Seven of these observed ICG accumulation in necrotic tumors, burn tissue and reperfusion lesions, suggesting that ICG has necrosis avidity.^9,24–29 In fact, intracellular uptake of ICG was directly observed in necrotic skin cells when imaged hours to days after dye administration due to cellular membrane dysfunction.³⁰ Loss of membrane integrity influenced ICG retention. Using colorectal carcinoma spheroids, ICG localized to the periphery of the spheroids outside their necrotic core after a one-hour incubation, but localized to the center of the necrotic spheroid core after incubations of three hours or more.³¹ In burns specifically, 24 hours after ICG administration, ICG accumulated throughout the entirety of necrotic burned mouse skin, whereas ICG penetration of thick desiccated burned human skin, which is much thicker than mouse skin, likely depends on the hydration state of the tissue to allow diffusion.²⁶ These two studies suggest that lack of sufficient time or variability in tissue conditions may impact diffusion of ICG into necrotic tissue subsequently impacting its retention in that tissue.

Studies that showed absence of fluorescence in necrotic tissue had experimental design variability. One study interpreted non-fluorescent regions within obvious tumor as areas of necrosis, but this finding was not confirmed histopathologically and imaging was performed 40 minutes instead of hours after ICG administration.³² Another study found that non-viable cells had a significantly lower fluorescence signal compared with viable cells, but cells were labelled with ICG prior to necrosis, making this finding inconsistent with clinical applications of SWIG imaging in which the pathology exists prior to ICG administration.³³ Overall, the timing of ICG administration critically impacts its retention in necrotic tissue.

ICG undergoes phagocytosis by the mononuclear phagocyte system (macrophages and monocytes) in inflammation

Twelve studies including cell culture, animal and human models in atherosclerosis, cancer, and infection co-localized ICG with various inflammatory cells, suggesting that ICG accumulates in inflammation regardless of the type of pathology.^{13,14,22,24,27,34–40} Some of the first studies on ICG retention in inflammation were in atherosclerosis, which demonstrated ICG retention localized within inflamed atherosclerotic lesions, and lack of ICG retention in non-lipid, non-inflamed plaques in vivo.^{14,22,36,37,41} More advanced atherosclerotic lesions, which had a higher percentage of inflammatory cells, had increased ICG fluorescence signal, whereas less severe atherosclerotic lesions exhibited less ICG fluorescent signal. Within areas of disrupted endothelium, ICG localized to zones of plaque macrophages, especially lipid-associated foam cells, lipid and intraplaque hemorrhage.^14,22,39 In vitro studies demonstrated ICG that bound to low-density lipoprotein and bovine serum albumin was internalized within human macrophages and foam cells in a concentration-dependent manner, indicating that phagocytosis by macrophages results in ICG retention (Figure 3B).^22,37

In cancer models including sarcoma, colorectal liver metastases and hepatocellular carcinoma (HCC) in which ICG fluorescence typically circumscribes the tumor, ICG co-localized to the areas containing inflammatory cells including granulocytes and macrophages.^27,38,40 In HCC, specifically, a close relationship between ICG accumulation and CD68 staining was observed.⁴⁰ However, this was disputed by another study in hepatocellular carcinoma that found no association between ICG accumulation and CD68-positive cells, and it is difficult to correlate ICG accumulation with macrophages in this setting because HCC cells also uptake ICG.⁴² Overall, further investigation needs to be done to evaluate ICG fluorescence corresponding to the inflammatory component of hepatocellular carcinoma.

The findings observed in atheroma and tumor-related inflammation are further supported by the work in peripheral human blood samples, in which phagocytic cell populations, specifically, monocytes and granulocytes, had stronger ICG fluorescence signals than other cell types found within blood after ICG administration.³⁴ These phagocytic cell types also demonstrated ICG localization with extranuclear vesicular structures (endosomes) when grown in culture, supporting the hypothesis that phagosomes uptake ICG through phagocytosis, a specific type of endocytosis (Figure 3B). The concentration of ICG, ambient temperature and duration of incubation all influenced the specificity of ICG binding to peripheral blood mononuclear cells including monocytes and macrophages, further supporting phagocytosis as a mechanism of ICG retention in inflammation.³⁵

Endocytosis contributes to cellular uptake of ICG in cancer cells

Tumors not only retain ICG due to phagocytosis by the inflammatory cells, but also due to uptake within cancer stromal cells. Twelve studies of all study designs observed ICG accumulation within the cytoplasm of hepatocellular carcinoma cells, suggesting a mechanism of ICG internalization.^{7,13,29,43–52} Tumor cell uptake of ICG does not appear to be specific to hepatocellular carcinoma. ICG fluorescence signal was observed within the cell membrane and cytoplasm of H460 non-small cell lung cancer cells incubated with ICG, which suggests that ICG inherently binds to the cell membrane before undergoing uptake within tumor cells.⁵³ Furthermore, intracellular uptake of ICG was observed to be directly proportional to the extracellular free-ICG concentration in melanoma and cervical cancer cells, an observation that specifically suggests endocytosis as a mechanism of ICG uptake in cancer cells (Figure 3C).⁵⁴

The contribution of endocytosis to ICG cellular uptake is further supported by the finding that ICG uptake is temperature-dependent.^18,31 More specifically, colorectal carcinoma spheroids treated at 4°C had a significantly lower ICG fluorescence intensity than those treated at 37°C.³¹ This, in conjunction with the finding that cellular uptake occurred after ICG binding to the cellular membrane, is specifically suggestive of clathrin-dependent endocytic uptake as a mechanism of ICG uptake.^18,31 Furthermore, multiple studies localized intracellular ICG fluorescence within the Golgi-endoplasmic reticulum system, then partially within mitochondria and lysosomes sequentially over time, further supporting endocytosis as a mechanism of cellular ICG uptake.^16,18,23 This finding could also imply that ICG undergoes autophagy by the autophagy lysosomal pathway, a major mechanism for degrading intracellular macromolecules. Finally, two studies observed both cytoplasmic and nuclear ICG fluorescence localization in hepatocellular carcinoma cells, suggesting that ICG can traffic through the nuclear pore complex.^46,55 It is unclear whether this occurs by passive diffusion or an active transport mechanism.

ICG is internalized into cancer cells via NTCP, OATP8 and OATP1B3 ion transporters

Specific influx ion transporters found in hepatocytes were shown to contribute to ICG retention in hepatocyte-derived tumors. Compared with HCC tissue that did not retain ICG, HCC tissue that retained ICG had significantly increased gene and protein expression levels of sodium taurocholate co-transporting polypeptide (NTCP) and organic-anion-transporting polypeptide 8 (OATP8), two ion transporters associated with portal uptake of ICG in hepatocytes (Figure 3C).²⁹ Differentiated HCC cells have preserved portal NTCP and OATP8 function, resulting in increased uptake of ICG, which behaves similarly to an organic anion, and results in the uniform fluorescence pattern observed grossly.²⁹ Another influx transporter, organic anion transporting polypeptide 1B3 (OATP1B3) was also increased in HCC cells that retained ICG.⁵⁶ Dedifferentiated HCC lose normal hepatocyte functionality and architecture. Poorly differentiated HCC cells had decreased OATP1B3 expression and poor uptake of ICG, thus explaining the partial or rim fluorescence observed grossly.^48,56

In non-hepatocellular cancers, ICG is similarly hypothesized to be transported from the extracellular space into the cytoplasm through OATP1B3 and NTCP ion transporters (Figure 3C).^57,58 ICG fluorescence signal was greater in human (MDA-MB-231) and murine (4T1) breast cancer cells engineered to express OATP1B3 compared with control cells without over-expressed OATP1B3.⁵⁷ Fluorescence microscopy confirmed intracellular ICG uptake in the OATP1B3-expressing cells, and lack-thereof in the control cells, suggesting that OATP1B3 promotes the cellular uptake of ICG.⁵⁷ ICG uptake in cultured cells and xenografted mice was studied after OATP1B3 was overexpressed in the cell membrane and nucleus of an HT-1808 sarcoma cell line.⁵⁹ ICG in the OATP1B3-expressing cells in both models had dose-dependent uptake into the cytoplasm.⁵⁹ These cells also had an ICG fluorescence signal greater than that of a control containing a sample of ICG at the treatment concentration, suggesting ICG uptake by OATP1B3 is an active or active-coupled process, rather than simple diffusion, which is consistent with the hypothesis of ICG uptake through ion transporters.⁵⁷

Similar studies were performed with NTCP.⁵⁸ ICG fluorescence intensity was higher in cells and tumors that expressed NTCP compared with controls that did not over-express NTCP. Moreover, NTCP-expressing cells had decreased ICG intensity after treatment with NTCP inhibitors, confirming the role of the NTCP ion transporter in ICG uptake.⁵⁸ Of note, induction of GLUT-1, a known transporter in many tumors, did not increase ICG uptake in HCC models, suggesting that GLUT-1 is not involved in ICG uptake.⁵¹

ICG is retained in hepatocyte-derived cells due to impaired efflux

Another mechanism for ICG retention is impaired ICG efflux. This hypothesis is based on the observation that ICG fluorescence imaging results of liver cancer were similar to those of delayed MRI imaging using a biliary-excreted contrast material.⁴⁷ Overall, damaged hepatocytes have impaired ICG cytoplasmic transport and biliary excretion (Figure 3D). Hepatocytes in cirrhotic livers had stronger ICG fluorescence intensity than those in non-cirrhotic livers, whereas non-cirrhotic livers had stronger ICG fluorescence signal within bile canaliculi, suggesting that impaired hepatocyte function results in ICG retention due to impaired biliary excretion.⁴⁶ In the incidence of liver dysfunction secondary to sepsis or severe systemic inflammation there is impaired canalicular biliary excretion of ICG, leading to its retention in hepatocytes.⁶⁰ Finally, microtubules have been shown to be involved in the cytoplasmic transport of ICG, likely via vesicles, before ICG is excreted into biliary canaliculi.⁶¹ Disorganization of the microtubular network due to liver ischemia contributes to impaired cytoplasmic transport of ICG.⁶¹ Cirrhosis, sepsis and ischemia all lead to loss of hepatocyte functionality, resulting in impaired excretion capacity and leading to ICG findings similar to those seen in liver cancers.

Liver tumors are unique because ICG is normally metabolized by hepatocytes and excreted through the biliary system. While this functionality is retained in well-differentiated HCC cancers, it becomes lost in dedifferentiated tumor cells. ICG was retained in the cytoplasm of well-differentiated cancer cells in tumors displaying uniform fluorescence. Conversely, in dedifferentiated tumors, ICG was absent in the cytoplasm of the HCC cells themselves, but rather present in the hepatocytes immediately surrounding the tumor resulting in a peripheral fluorescence pattern.⁵² The various ICG cellular retention patterns described as peripheral versus uniform fluorescence is explained by the concept that there is impaired excretion in the cells with ICG retention, which corresponds to the differentiated status of the cancer. Studies ascribe impaired ICG excretion to disrupted biliary function in the surrounding normal liver tissue from compression by the tumor, thus causing slow biliary flow or stasis.^{11,38,40,42,52,62–65} This statement is based on the observation of multiple studies that ICG is retained in pseudoglands, the canalicular side of the cancer cell cytoplasm or intercellular spaces of bile canaliculi.^{7,29,44,45,47,52,56} Additionally, ICG was retained around hepatocytes with CK7-positivity, a marker of ductular transformation in immature hepatocytes, bile duct injury and biliary stasis.⁴² These findings support the hypothesis that ICG is excreted to extracellular spaces including pseudoglands, where it is retained due to disrupted lymphatic or biliary efflux.

Bile efflux can be disrupted not only due to morphological changes from tumor compression and mass effect, but also cellular de-differentiation and loss of normal efflux transporters (Figure 3D). ICG was observed in the pseudoglands or intercellular spaces of HCC cells abundantly expressing the efflux transporter multidrug resistance p-glycoprotein (MDR)-3 compared with cancer cells that had low ICG retention.⁵⁶ Similarly, HCC cells with uniform fluorescence had increased gene and protein expression levels of the efflux transporter multidrug resistance-associated protein 2 (MRP2) compared with HCC tissue exhibiting peripheral fluorescence.²⁹ Increased efflux transporter expression may allow for increased ICG excretion via the biliary system, which protects the dye from intracellular degradative systems such as lysosomal autophagy and the ubiquitin-proteasome system. A similar phenomenon was observed in immature, reactive hepatocytes surrounding tumors with peripheral fluorescence.^11,52,62 Overall, there is evidence that ICG is retained in tumor cells and immature, reactive hepatocytes due to impaired efflux pathways from lack of specific cell transporters, ^{11,29,52,56,62} as well as biliary stasis.^{11,38,40,42,52,62–65} This phenomenon contributes to non-specific ICG retention.

DISCUSSION

In clinical disease processes causing vascular permeability, ICG is retained in pathological tissue, making it feasible for SWIG imaging to identify a diseased state. From the longstanding history of ICGA, and the more recent investigations of SWIG, it is clear that there are several clinical factors that influence SWIG fluorescence overall: timing of imaging after ICG administration, ICG dose, imaging device, ambient lighting, tissue type, overall inflammatory state, and patient comorbidities (Figure 4). SWIG imaging is performed after ICG has been cleared in normal tissue yet remains within the diseased tissue. This requires a delay of at least several hours after ICG administration. SWIG also requires high dose ICG administration. Lower doses result in insufficient SWIG signal-to-background fluorescence, likely due to less overall dye penetrating the diseased tissue. Similarly, patient factors such as cardiac output, overall metabolic state and perfusion defects can influence the timing and overall interaction between ICG dye and pathologic target of interest, and thus affect SWIG.

Figure 4.

Imaging and patient factors that influence second window indocyanine green (SWIG) fluorescence imaging. Created with Biorender.com.

The broad purpose of this scoping review was to synthesize what is known about the mechanism of ICG retention at the cellular level to better understand the clinical nuances of SWIG imaging and identify critical gaps in our knowledge to serve as direction for future study. In our review, 183 manuscripts that were otherwise eligible for inclusion were excluded because the study was justified solely based on the EPR effect without inclusion of original mechanistic details in the results. We identified several key mechanistic features involved in ICG accumulation in diseased tissue (Table 2 and Supplemental Table 2).

Our findings suggest that ICG retention in pathological tissues including solid tumors and necrosis is overall dependent on a specific cell’s ability to bind and/or internalize ICG via endocytosis or ion transporters (Figure 3), which implies that the mechanism of ICG retention in tumors and non-tumor inflammation may not be synonymous. Cellular binding of ICG may be non-specific, but cellular uptake seems to involve specific cell populations. The studied ion transporters responsible for ICG uptake are found in healthy hepatocytes, and tumor cells derived from hepatocytes and non-hepatocytes. Phagosomes and some cancer cells appear to utilize endocytosis for cellular ICG internalization, but further investigation is needed to define these specific cell populations and timing of ICG uptake. Furthermore, ICG that does not undergo cellular uptake but rather remains within the interstitium likely undergoes phagocytosis by the mononuclear phagocyte system, ultimately leading to its breakdown.

Regarding the use of ICG to identify necrotic tissue, it has been preliminarily demonstrated that ICG binds to exposed phospholipids on damaged cellular membranes (Figure 3A).²⁴ We hypothesize that ICG extravasates through permeable vessels and binds to necrotic cell membranes in a diffusion dependent manner in which the necrotic tissue closest to capillaries has the highest ICG binding. We hypothesize that the stage of membrane dysfunction due to cellular necrosis influences whether ICG is internalized. Cells earlier in the necrosis pathway will have altered plasma and organelle membranes, permitting possible uptake of ICG. Cells in a later stage of the necrosis pathway will have undergone disruption then breakdown of their organelles, resulting in an inability to internalize ICG. Further studies on the progression of necrosis in relation to ICG binding and uptake, as well as understanding the natural history of necrosis within the specific disease process of interest are imperative to optimization of SWIG technology.

One limitation of this study is the exclusion of manuscripts written in languages other than English. However, English is the lingua franca of scientific literature with more than 90% of indexed scientific articles published in this language.⁶⁶ Because of this, and the nature of this manuscript as a scoping mechanism rather than a meta-analysis, this limitation should not significantly impact the overall findings of this work.

While ICG retention within tumor and non-tumor tissues may involve distinct mechanisms, overall, these findings support that SWIG imaging requires optimization through specific dosing and timing of image acquisition to localize the pathology of interest. Future SWIG investigation should involve disease-specific applications involving both clinical and cellular studies.

CONCLUSION

This scoping review synthesized what is known about the mechanism of ICG retention at the cellular level to better understand the clinical nuances of ICG imaging and identify critical gaps in our knowledge to serve as direction for future study. We identified key mechanistic features involved in ICG accumulation in diseased tissue that demonstrate that the mechanism of ICG retention is dependent on the disease process. Our study also provides a new connection on how inflammatory infiltrate influences ICG fluorescence imaging. These findings are significant because they add to our understanding of how fluorescence-guided surgery could be optimized based on the pathology of interest via specific ICG dosing and timing of image acquisition.

Supplementary Material

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Supplemental Table 1. All included studies.

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Supplemental Table 2. Included studies on delayed ICG fluorescence imaging of cancer, inflammation and necrosis with disease process, ICG dose, imaging timing, ICG fluorescence signal localization and overall significance of imaging findings.

TWO SENTENCE ARTICLE SUMMARY.

A scoping review was performed identifying key mechanistic features involved in ICG accumulation in diseased tissue to elucidate the mechanism of ICG retention in tumors, inflammation and necrosis. The importance of this review is that mechanisms of delayed imaging for fluorescence-guided surgery are not fully elucidated and appear to be based on the pathology of interest, specific ICG dosing, and timing of image acquisition.