TIME for Bugs: The Immune Microenvironment and Microbes in Precancer

Carcinogenesis is typically defined as the process by which normal cells transform into tumor cells through acquiring characteristics that confer a survival advantage. In addition, the term also represents a coevolution of the immediate cellular environment, including the immune infiltrate, surrounding microbes, stroma, and vasculature. In many tumor types, the progression from normal tissue to invasive malignancy has been extensively studied and characterized, detailing the genomic, transcriptomic, proteomic, and epigenomic changes within tumor cells across the spectrum of carcinogenesis. Further, many studies exist on describing the tumor immune microenvironment (TIME) in established cancer, as well as microbial changes associated with advanced cancer and therapy response. Fewer studies, however, have documented the parallel changes within the immune microenvironment or microbiome during carcinogenesis, which simultaneously progress towards an overwhelmingly immunosuppressive and dysbiotic phenotype. This coevolution is a dynamic process with intricate communication between the developing tumor cells, the local and systemic immune system, as well as components of the gut and regional tissue microbiome (graphical depiction in Fig. 1).

This review focuses on the contribution of local and systemic changes to the immune response and microbiome in premalignancy, with sections addressing (i) tumor–immune crosstalk during carcinogenesis, (ii) evolution of immune microenvironment during premalignancy, (iii) the interplay of the immune milieu and microbiome in precancerous lesions, (iv) the influence of risk factors and risk-reducing interventions, (v) translational research opportunities for defining the immune microenvironment in premalignancy, and (vi) future research opportunities. We review the promising application of immune- and microbiome-targeting therapies for cancer prevention as well as point out critical knowledge gaps that remain.

Oncogenic pathway activation – through gain-of-function alterations of oncogenes or loss-of-function of tumor suppressor genes – has historically defined the earliest stages of cancer development. Driver mutations in oncogenes and tumor suppressor genes were initially assumed to modulate cell-intrinsic processes only. However, these factors dramatically affect cell-extrinsic processes, including the antitumor immune response. Cross-talk between driver mutations in tumor cells and the immune environment occurs with multiple oncogenic pathways, including Myc, β-catenin, PTEN, and many others. Myc and its related signaling pathways are among the most commonly mutated oncogenes in human cancer, driving uncontrolled cell division. Myc, however, also regulates the immune microenvironment via reducing antigen presentation (decreased MHC I and II expression), limiting immune-mediated killing by macrophages and natural killer (NK) cells, and preventing T-cell infiltration (1, 2). Myc activation in preclinical pancreatic cancer models also induces rapid recruitment of mast cells, which are necessary for tumor growth (3). β-Catenin signaling is disrupted in many tumor types, including melanoma and colorectal cancer. Alongside its roles in cell proliferation and migration, β-catenin signaling also represses immune cell activation by preventing infiltration of BATF⁺ dendritic cells (DC), which are critical in priming naïve T cells, and by inhibiting T-cell recruitment (2). A final example of crosstalk between driver mutations in tumor cells and the immediate immune environment is that of PTEN loss, which occurs early in the development of many tumor types. In preclinical prostate cancer models, PTEN loss results in MDSC recruitment and the shifting of cytokines and inflammatory modulators towards an immunosuppressive phenotype (4). PTEN loss can promote NF-κB activation during pancreatic ductal adenocarcinoma (PDAC) development (5). PTEN loss also modifies the composition of infiltrating immune cells in the pretumor microenvironment, including increases in protumorigenic macrophages in liver cancer development (6) and reductions of CD8⁺ T cells in melanoma (7).

Accumulation of somatic mutations and DNA copy-number alterations during tumor development also affects antitumor immune responses. Point and frameshift mutations are a primary source of tumor neoantigens, which are recognized by the host immune system and can facilitate immune-mediated tumor cell killing. However, progressive chromosomal aberrations and aneuploidy is associated with decreased adaptive and innate immune cell infiltration, decreased ratios of CD8⁺:CD4⁺ T cells and M1:M2 macrophages, and decreased expression of cytolytic proteins and IFN signaling (8).

Preclinical work continues to identify novel ways in which driver mutations and chromosomal aberrations impact antitumor immune activity. Coculture systems of tumor cells and isolated CD8⁺ T cells have been employed to identify novel genes that confer resistance of tumor cells to cytotoxic T cells in vitro (9). In addition, comparisons of immunodeficient versus wild-type mice have identified many novel tumor suppressor genes specific to modulation of immune activity (10). The sheer number of novel genetic elements identified in these studies suggests that our view of the coevolution of tumor cells and the immune system remains quite naïve, necessitating additional studies to increase understanding of the sophisticated crosstalk between tumor cells and immune microenvironment during cancer development.

Although oncogenic signaling pathways generally stimulate immune activation early in malignant transformation, the transition to evasion of antitumor immune response is a Hallmark of Cancer (11). Translational studies have demonstrated evidence of both immune activation and evasion at the premalignant state, prior to invasion.

In both lung and oral squamous cell carcinoma (SCC), detailed transcriptional and histologic analyses along the carcinogenesis spectrum have allowed longitudinal assessment of immune involvement, revealing marked changes in immune activity prior to tumor invasion. Neutrophils and naïve T cells dominate the immune microenvironment early in lung SCC development, in association with high cancer neoantigen expression (12). High-grade lesions exhibit increased T-cell activation prior to invasion, then express markers of T-cell exhaustion during the transition from in situ disease to invasive carcinoma. Moreover, expression of antitumor immune response genes decreases during epithelial–mesenchymal transition (EMT; ref. 12). In oral SCC, early lesions exhibit high levels of tumor-infiltrating lymphocytes. Some of these early lesions spontaneously regress, suggesting an active antitumor immune response. Lesions that progress to oral SCC, rather, exhibit increases in immune checkpoint expression and in infiltrating immunosuppressive cell types, including MDSCs, M2 macrophages, and regulatory T cells (Tregs ref. 13).

Similarly, progressive downregulation of immune activation pathways alongside upregulation of immunosuppressive pathways occurs during adenocarcinoma development. Parallel histologic studies exhibit decreased infiltration of cytotoxic and Th cells and increased infiltration of Tregs during the progression to preinvasive and early lung adenocarcinoma (14). A longitudinal cohort study of patients with the premalignant breast cancer lesion ductal carcinoma in situ (DCIS) determined that features associated with immune activation, such as CD4⁺ T-cell and mast cell infiltration, were observed primarily in DCIS and not invasive breast cancer lesions (15).

Evidence of decreased immune activation with concurrent increases in immunosuppressive factors at the earliest stages of carcinogenesis, prior to overt invasion, implies that immune activation and evasion occur in the premalignant state. These data suggest that earlier immunomodulatory treatment strategies might be not only feasible but potentially more efficacious than in the more deranged immune microenvironment of advanced malignancy. Further work in understanding the immune response in premalignant lesions is critical to address these gaps in knowledge.

There is an ever-growing appreciation for the role of the microbiome in cancer – from the earliest steps in carcinogenesis all the way to metastasis. We focus here on the beginning of tumor formation and outline evidence for a continuum of microbial changes that parallel transformation of tumor cells and the tumor immune microenvironment. Whereas various viruses and bacteria have been elucidated as causative agents in multiple cancers (e.g., H. pylori-associated gastric cancers and human papillomavirus (HPV)-associated cervical, oral, and anal cancers), it is becoming increasingly evident that commensal microbiota further shape tumorigenesis and the antitumor immune response. As an example, although H. pylori is associated with gastric cancer, H. pylori infection does not cause cancer in most patients, suggesting that other factors modify its carcinogenic activities. H. pylori infection is associated with gastric dysbiosis, specifically with decreased abundance and variety of other microbes present in the uninfected state (16). Moreover, in preclinical models, germ-free mice infected with H. pylori develop gastric intraepithelial neoplasia less often and more slowly than conventional mice or mice with a restricted microbiome (16). These data suggest that components of the gastric microbiome other than H. pylori may synergize in gastric carcinogenesis. Similarly, while HPV is a known causative agent for both cervical intraepithelial neoplasia (CIN) and cervical cancer, only a small fraction of infected women has persistent HPV infection and develops lesions. The cervicovaginal microbiome may alter the ability of HPV to persist and cause infection. In fact, a signature of cervicovaginal microbiome consisting of increased diversity of microbiota, increased Gardnerella spp., and reduced Lactobacillus spp. is associated with development of cervical premalignant lesions (17, 18). Furthermore, researchers identified a bacterial signature in the perianal region linked with higher-risk HPV-related precancerous anal intraepithelial neoplastic lesions more likely to progress to carcinoma (19).

Microbes influence cancer development via both direct and indirect roles. The premalignant tissue-resident bacteria have been explored in greatest depth for gastrointestinal cancers. In colorectal cancer, differences in the microbiome are associated with serrated versus adenomatous colorectal carcinogenic pathway. Increases in microbial richness, diversity, and specific species are apparent in adenomatous lesions compared with normal colonic tissue (20). Biofilms, associated with bacterial overgrowth and invasion, are found in right-sided colonic tumors, premalignant adenomas, and adjacent normal tissue, suggesting that they may play a role in early-tumor formation (21). Patients suffering from chronic gastritis or other gastric preneoplastic lesions have altered gastric pH, which facilitates survival of some oral bacteria and other intestinal commensals, particularly nitrosating bacteria, which can have genotoxic effects (22). Indeed, the microbiome of patients with premalignant gastric lesions, when transplanted into germ-free mice, induced gastric dysplasia (23). Gut microbes even exhibit the ability to hijack hormone signaling, where gut dysbiosis induces changes to estrogen metabolism (24) and production of androgens during prostate cancer development (25), which may tune cancer development both locally or distantly. Further, cross-talk between the immune microenvironment and microbiome facilitate oncogenesis. In PDAC, an influx of bacterial colonization from the stomach to the pancreas reprograms the developing tumor microenvironment towards an immunosuppressive phenotype (26). In additon, intratumoral bacteria, which are present inside tumor cells and local immune cells, vary among tumor types (including those outside the gut) in both diversity and metabolic profile, suggesting their roles in cancer–immune signaling (27). However, comprehensive analyses of intratumor microbiomes across tissue types are lacking for premalignant lesions, especially those beyond the digestive system.

Although the role of the fungal microbiome is not yet well-defined, very recent studies demonstrate its potential to influence carcinogenesis across multiple cancer types. Large pan-cancer sequencing studies identified fungal signatures associated with gastrointestinal cancer development (28, 29). In pancreatic cancer, activation of the fungal complement cascade via mannose-binding lectin is required for oncogenesis (30). Also in pancreatic cancer, oncogenic Kras has been found to increase IL33 expression, which is dependent on the intratumoral mycobiome and can be modulated via antifungal treatment (31). In oral premalignant lesions, those associated with Candidal infection were more likely to progress to oral SCC, and clearance of Candida leads to regression in some patients. However, whether Candida causes or is merely associated with oral premalignancy is not known and may depend on other factors, such as immune activity and general dysbiosis (32). Additional studies across the cancer development continuum are necessary to decipher the distinct roles of commensal versus pathogenic fungi in carcinogenesis.

Commensal microbes, the symbiotic bacteria that normally play a role in host defense and digestion, have the capacity to influence cancer initiation systemically as well as locally, via mechanisms distinct from intratumoral bacteria. Commensal microbes secrete factors that regulate hormone-producing cells and immune cells. For example, microbe-mediated hormone release occurs in the gut, whereby commensals influence the release of various gastrointestinal hormones (including CCK, PYY, GLP-1, GIP, and 5-HT) by enteroendocrine cells, which have systemic effects on insulin sensitivity, glucose tolerance, and other metabolic functions (reviewed in ref. 33). Moreover, the gut microbiome influences the immune system by either signaling to local immune cells within the lamina propria or nearby lymphoid tissues (reviewed in ref. 34), or by releasing metabolites (such as short chain fatty acids, amino acids, and bile acids) into the circulation. For example, short-chain fatty acids activate peripheral Tregs (35–37) with mainly immunosuppressive effects. Distinct systemic cancer-related microbial signatures have also been identified in blood versus tissue signature studies from The Cancer Genome Atlas (TCGA), and in fact revealed the utility of liquid biopsy of microbial signatures for cancer detection (38). However, a knowledge gap still exists on the influence of commensals in cancer development specifically, as well as distinctions between the impact of commensal versus intratumoral microbes on cancer development beyond those in and around the digestive system. These and aforementioned data signify the influence of the microbiome on early-stages of carcinogenesis and support the use of microbial signatures as biomarkers for high-risk lesions. Moreover, these findings indicate the potential utility for microbial intervention in cancer interception.

Thus far, strategies to alter the anti-tumor immune response or cancer-associated dysbiosis have typically been employed after diagnosis of overt malignant disease, often in the setting of advanced or even metastatic cancer. Evidence of the immune and microbial influence in early carcinogenesis underscores the importance of evaluating the influence of risk factors and risk-reducing interventions in modulating the immune microenvironment and microbiome to reverse disease progression. Here, we will specifically focus on obesity, inflammation, and hereditary cancer risk.

Obesity is a multifactorial disorder, characterized by chronic systemic and local inflammation, metabolic dysfunction, and microbial dysbiosis, all of which contribute to immunosuppression during carcinogenesis. For example, in preclinical models, obesity promotes inflammation and immunosuppression within the liver, permitting obesity-related hepatocellular carcinogenesis. Obesity-induced gut microbial COX2 signaling causes PGE₂ production in hepatic stellate cells, which suppresses antitumor immunity during carcinogenesis via modulation of local cytokine expression (39). The inflammatory state in obesity promotes leakage of microbial metabolites into the portal circulation (gut–liver axis), further amplifying inflammation within the liver. Obesity-related gut dysbiosis has been implicated in other cancer development processes, including in endometrial cancer, where obesity- and menopause-related gut dysbiosis affect estrogen metabolism and promote recruitment of Tregs to the endometrium (40). Obesity similarly affects the vaginal and uterine microbiome (41), where dysbiosis, including reduced diversity and changes to the dominant microbial species, has been observed early during endometrial carcinogenesis (42). Obesity impacts the immune system in endometrial cancer risk also. Obese women who prospectively underwent bariatric surgery for weight loss (with predicted endometrial cancer risk reduction) subsequently had decreased systemic inflammatory biomarkers (CRP, IL6) and increased endometrial infiltration of CD8⁺ T cells (43). Finally, obesity has been shown to affect not only the proportion of immune cell types in the microenvironment but also their function. Secondary to metabolic dysfunction in obesity, competition for lipids in the immune microenvironment causes CD8⁺ T-cell dysfunction (44).

Inflammation also modulates cancer development via the immune microenvironment in other chronic conditions. In inflammatory bowel disease (IBD), chronic inflammation induces both dysbiosis and an immunosuppressive microenvironment. Inflammatory insults to intestinal barrier integrity (45) cause an influx of microbes, often from a dysbiotic background, into tissues at risk of cancer development (46). Chronic inflammation in ulcerative colitis is characterized by increased TNFα signaling (47), IL6-dependent STAT3 activation (48, 49), and Th17 cell infiltration (50), all of which contribute to proinflammatory, protumorigenic signaling. Furthermore, activation of innate immunity plays a dual role in colitis-associated carcinogenesis: while activation of innate immunity has been shown to protect against colitis-associated carcinogenesis, it also contributes further to proinflammatory, protumorigenic signaling within ulcerative colitis lesions (51). In Barrett's esophagus, premalignant lesions characterized by chronic inflammation, increased IL6 and CXCL8 production alongside higher numbers of M2 macrophages, pro-B cells, and eosinophils are associated with progression to esophageal adenocarcinoma (52). Controlling inflammation via surgical or pharmacologic intervention in these chronic diseases can prevent malignancy (53, 54).

Genetic predisposition for cancer is a nonmodifiable risk factor yet represents a unique opportunity for immune-based cancer interception strategies. Lynch syndrome, or hereditary nonpolyposis colorectal cancer syndrome (HNPCC), is caused by inherited loss of DNA mismatch repair genes. Resultant accumulation of mutations due to defective DNA repair increases the risk for developing colorectal, endometrial, and ovarian cancers (55). Hypermutability also causes production of neoantigens, frame-shift peptides recognized by the immune system (56). Importantly, immune reactivity to cancer-related neoantigens precedes evidence of even histologic premalignant (adenomatous polyp) transformation (57). Intervention at early stages in carcinogenesis has the potential to prevent cancer development. The NSAID naproxen exhibits efficacy against colorectal cancer development in part due to its effects on the immune system, by the activation of T and B cells (without altering the number of infiltrating immune cells) (58). These findings have led to increased interest in developing precision immune interception strategies against cancer development in patients with Lynch syndrome, including neoantigen-targeted vaccines (59).

Leveraging the precancerous immune and microbial microenvironment against cancer development has the potential to revolutionize cancer prevention strategies, especially for patients with known risk factors, such as obesity, chronic inflammatory conditions or infections, and genetic predisposition. Immuno-preventive strategies, including vaccination and immunotherapy, are discussed elsewhere in this series, but opportunities also exist for lifestyle modification in harnessing the potential of the immune and microbial microenvironment against cancer development. Many studies focus on the modulatory effects of dietary changes on antitumor immune activity and the microbiome in patients that already have invasive cancer, where a high-fiber diet has been shown to enhance response to immune checkpoint blockade via modulation of the gut microbiome (60). Preclinical studies have demonstrated the utility of probiotic use for colorectal cancer prevention, where various probiotics (Clostridium butyricum, Bacillus subtilis, Lactobacillus rhamnosus, or a cocktail of Lactobacillus acidophilus, Bifidobacteria bifidum, and Bifidobacteria infantum) show potential prophylactic effect against an inflammatory model of colorectal cancer development (61–63). Pre- and pro-biotic supplementation with a preparation of oligofructose-enriched inulin, Lactobacillus rhamnosus GG, and Bifidobacterium lactis Bb12, also decreased genotoxicity and reduced IL2 production by peripheral blood mononuclear cells in patients with precancerous colonic lesions in a randomized controlled trial (64). However, in the setting of cancer recurrence, dietary fiber (wheat bran) and/or probiotic supplementation (Lactobacillus casei) does not appear to have the same protective effect (65), supporting the notion that lifestyle modification in the precancerous stage may have the most significant impact.

Several clinical trials evaluating the impact of microbiome modulation on cancer prevention have launched. Such trials include the effect of dietary interventions on commensal microbiome and subsequent impact on colorectal cancer prevention in patients at increased risk due to obesity (NCT02843425, NCT03582306) or lung cancer in patients at increased risk due to significant smoking history (NCT04267874). As an example, the ongoing BE GONE trial (66) implements the addition of one cup of canned beans per day to each participant’s typical diet as a preventive approach for overweight and obese patients with a history of colorectal polyps or cancer (primary objective is evaluation of gut microbiome plus blood adipokine and cytokine biomarkers). This trial builds from both human and rodent models that show anti-inflammatory and chemopreventive influence of beans (67–69), including a trial (NCT01929122) which showed rice bran or navy bean interventions increase microbial diversity and alter microbiome composition after 28 days (70). An alternative approach evaluated providing participants with frozen dark leafy green vegetables in 1 cup “doses” daily in addition to their typical diet (NCT03582306), and has demonstrated feasibility for accrual, retention, and acceptability (primary outcomes), but secondary outcomes (microbiome composition, DNA damage, and inflammatory biomarker studies) are pending. In the BE WELL lung cancer trial (NCT04267874) the effects of black raspberry nectar powder on microbiome and inflammation markers, but results have not yet been reported. Other trials evaluating microbiome modulation with non-dietary interventions include the use of metformin for prevention of cancer in patients with oral precancerous lesions (NCT02581137) and omega-3 polyunsaturated fatty acid supplementation in participants with a history of colorectal adenoma (NCT04216251). These trials, while providing valuable clinical information on the impact of targeting the microenvironment for cancer prevention, will also have the added opportunities for tissue-based studies to increase understanding of the determinants for immune- and microbiome-based cancer interception.

Broad interest and enthusiasm for cancer immunotherapy research has not yet transferred to the premalignant setting, which may be more tractable for immune-based interventions. Cancers with a well-characterized preneoplastic lesion, accessible tissue location, and well-defined risk factors are an ideal setting to define the role of immune response and immunoprevention for cancer interception. In the past, limited tissue availability, small lesion size, and substantial regional heterogeneity have proven challenging for translational research in the premalignant setting. While local heterogeneity in immune infiltrate and oncogenic signaling can drive either regression or progression of precancerous lesions, it has been difficult to define these interactions. Recently, however, methodologic advances provide ample opportunities in this realm. Analysis of regional variation had not previously benefited from deep profiling due to platforms that lacked spatial resolution. However, novel methods, such as spatial transcriptomics and/or proteomics, allow dissection of regional crosstalk between oncogenic signaling pathways and the immune microenvironment, which is a particularly powerful approach for small or otherwise limited precursor lesions. Furthermore, evaluation of regional microbes and local immune signaling with spatial resolution will enable interrogation of the crosstalk between key signaling pathways that drive the progression or regression of premalignant lesions. Advances in methodology for assessing the immune and microbiome microenvironment of precancerous lesions will be complemented by novel screening techniques for precursor lesions, including liquid biopsy (its application to cancer prevention reviewed in ref. 71), nanoparticle-directed imaging modalities (reviewed in ref. 72), and microfluidic nanochip-based detection of tumor-specific exosomes (73). These novel screening techniques will further advance cancer interception by allowing for earlier detection. Identification of lesions early in carcinogenesis, along with insight on the composition of the pretumor microenvironment, will inform the utility of immune and microbiome targeting for cancer interception.

Traditional approaches to cancer interception have attempted to target specific oncogenic signaling pathways but have had limited uptake in at-risk populations. New cancer interception strategies focused on the immune microenvironment and the microbiome may enable broader applications due to the potential for intermittent interventions to support antitumor immune response and relevance across multiple tissue/tumor types. Importantly, enhanced pharmacologic approaches are being partnered with cancer interception agents (overview included in ref. 74), which could support efficacy of local immune modulation. Alternatively, multi-organ cancer interception strategies with potential to improve multiple morbidities associated with cancer risk factors (such as obesity or chronic inflammation) will also have the greatest likelihood of a favorable cost/benefit ratio. While dietary interventions have been studied for cancer prevention for decades, the recent understanding of the role of fiber and other dietary modifications in modifying the gut microbiome and response to immunotherapy (60, 75, 76) provides a new paradigm, including new potential biomarkers and routes for modulation. Given the well-recognized role of the microbiome in cancer development and progression, along with many remaining knowledge gaps, future study designs and specimen collection procedures must incorporate appropriate techniques to enable analysis of the tissue and gut microbiome with necessary rigor. Although earlier large-scale studies leveraged existing specimens and sequencing data to interrogate the intratumoral microbiome in an opportunistic manner, prospective sampling of gut and tissue microbiome will enable critical advances, particularly for premalignant conditions and at-risk patient populations. As mentioned above, spatial biology techniques will enable powerful opportunities for integration of microbiome analyses into multi-platform profiling studies.

J.A. Wargo reports personal fees from Imedex, Dava Oncology, Omniprex, Illumina, Gilead, PeerView, Physician Education Resource, MedImmune, Exelixis, and personal fees from Bristol Myers Squibb outside the submitted work; in addition, J.A. Wargo has a patent for PCT/US17/53.717 issued and has served as a consultant/advisory board member for Roche/Genentech, Novartis, AstraZeneca, GlaxoSmithKline, Bristol Myers Squibb, Micronoma, OSE therapeutics, Merck, and Everimmune. J.A. Wargo received stock options from Micronoma and OSE therapeutics. No disclosures were reported by the other authors.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the funders.

M.B. Bowen was supported by a predoctoral fellowship from the Cancer Prevention and Research Institute of Texas (RP210042). All authors were supported by the Cancer Center Support Grant at MD Anderson Cancer Center (CA016672).