Microbiome bacterial influencers of host immunity and response to immunotherapy.

The gut microbiota influences anti-tumor immunity and can induce or inhibit response to immune checkpoint inhibitors (ICIs). Therefore, microbiome features are being studied as predictive/prognostic biomarkers of patient response to ICIs, and microbiome-based interventions are attractive adjuvant treatments in combination with ICIs. Specific gut-resident bacteria can influence the effectiveness of immunotherapy; however, the mechanism of action on how these bacteria affect anti-tumor immunity and response to ICIs is not fully understood. Nevertheless, early bacterial-based therapeutic strategies have demonstrated that targeting the gut microbiome through various methods can enhance the effectiveness of ICIs, resulting in improved clinical responses in patients with a diverse range of cancers. Therefore, understanding the microbiota-driven mechanisms of response to immunotherapy can augment the success of these interventions, particularly in patients with treatment-refractory cancers.

Dissecting mechanisms of fecal microbiota transplantation efficacy in disease.

Fecal microbiota transplantation (FMT) has emerged as an alternative or adjunct experimental therapy for microbiome-associated diseases following its success in the treatment of recurrent Clostridioides difficile infections (rCDIs). However, the mechanisms of action involved remain relatively unknown. The term 'dysbiosis' has been used to describe microbial imbalances in relation to disease, but this traditional definition fails to consider the complex cross-feeding networks that define the stability of the microbiome. Emerging research transitions toward the targeted restoration of microbial functional networks in treating different diseases. In this review, we explore potential mechanisms responsible for the efficacy of FMT and future therapeutic applications, while revisiting definitions of 'dysbiosis' in favor of functional network restoration in rCDI, inflammatory bowel diseases (IBDs), metabolic diseases, and cancer.

Fecal microbiota transplantation plus anti-PD-1 immunotherapy in advanced melanoma: a phase I trial.

Fecal microbiota transplantation (FMT) represents a potential strategy to overcome resistance to immune checkpoint inhibitors in patients with refractory melanoma; however, the role of FMT in first-line treatment settings has not been evaluated. We conducted a multicenter phase I trial combining healthy donor FMT with the PD-1 inhibitors nivolumab or pembrolizumab in 20 previously untreated patients with advanced melanoma. The primary end point was safety. No grade 3 adverse events were reported from FMT alone. Five patients (25%) experienced grade 3 immune-related adverse events from combination therapy. Key secondary end points were objective response rate, changes in gut microbiome composition and systemic immune and metabolomics analyses. The objective response rate was 65% (13 of 20), including four (20%) complete responses. Longitudinal microbiome profiling revealed that all patients engrafted strains from their respective donors; however, the acquired similarity between donor and patient microbiomes only increased over time in responders. Responders experienced an enrichment of immunogenic and a loss of deleterious bacteria following FMT. Avatar mouse models confirmed the role of healthy donor feces in increasing anti-PD-1 efficacy. Our results show that FMT from healthy donors is safe in the first-line setting and warrants further investigation in combination with immune checkpoint inhibitors. ClinicalTrials.gov identifier NCT03772899 .

IL-12 and IL-27 Promote CD39 Expression on CD8+ T Cells and Differentially Regulate the CD39+CD8+ T Cell Phenotype.

Tumor-specific CD8+ T cells are critical components of antitumor immunity; however, factors that modulate their phenotype and function have not been completely elucidated. Cytokines IL-12 and IL-27 have recognized roles in promoting CD8+ T cells' effector function and mediated antitumor responses. Tumor-specific CD8+ tumor-infiltrating lymphocytes (TILs) can be identified based on surface expression of CD39, whereas bystander CD8+ TILs do not express this enzyme. It is currently unclear how and why tumor-specific CD8+ T cells uniquely express CD39. Given the important roles of IL-12 and IL-27 in promoting CD8+ T cell functionality, we investigated whether these cytokines could modulate CD39 expression on these cells. Using in vitro stimulation assays, we identified that murine splenic CD8+ T cells differentially upregulate CD39 in the presence of IL-12 and IL-27. Subsequently, we assessed the exhaustion profile of IL-12- and IL-27-induced CD39+CD8+ T cells. Despite the greatest frequency of exhausted CD39+CD8+ T cells after activation with IL-12, as demonstrated by the coexpression of TIM-3+PD-1+LAG-3+ and reduced degranulation capacity, these cells retained the ability to produce IFN-γ. IL-27-induced CD39+CD8+ T cells expressed PD-1 but did not exhibit a terminally exhausted phenotype. IL-27 was able to attenuate IL-12-mediated inhibitory receptor expression on CD39+CD8+ T cells but did not rescue degranulation ability. Using an immunogenic neuro-2a mouse model, inhibiting IL-12 activity reduced CD39+CD8+ TIL frequency compared with controls without changing the overall CD8+ TIL frequency. These results provide insight into immune regulators of CD39 expression on CD8+ T cells and further highlight the differential impact of CD39-inducing factors on the phenotype and effector functions of CD8+ T cells.

The Role of Microbiota-Derived Vitamins in Immune Homeostasis and Enhancing Cancer Immunotherapy.

Not all cancer patients who receive immunotherapy respond positively and emerging evidence suggests that the gut microbiota may be linked to treatment efficacy. Though mechanisms of microbial contributions to the immune response have been postulated, one likely function is the supply of basic co-factors to the host including selected vitamins. Bacteria, fungi, and plants can produce their own vitamins, whereas humans primarily obtain vitamins from exogenous sources, yet despite the significance of microbial-derived vitamins as crucial immune system modulators, the microbiota is an overlooked source of these nutrients in humans. Microbial-derived vitamins are often shared by gut bacteria, stabilizing bioenergetic pathways amongst microbial communities. Compositional changes in gut microbiota can affect metabolic pathways that alter immune function. Similarly, the immune system plays a pivotal role in maintaining the gut microbiota, which parenthetically affects vitamin biosynthesis. Here we elucidate the immune-interactive mechanisms underlying the effects of these microbially derived vitamins and how they can potentially enhance the activity of immunotherapies in cancer.

Improved MAIT cell functions following fecal microbiota transplantation for metastatic renal cell carcinoma.

Strategies to modify the gut microbiome in cancer patients using fecal microbiota transplantation (FMT) have gained momentum as a therapeutic intervention. However, how FMT impacts innate-like, antimicrobial T lymphocytes is unclear. In this study, we assessed peripheral blood (PB) mucosa-associated invariant T (MAIT) cell frequencies and functions in patients with metastatic renal cell carcinoma (mRCC) before and seven days after they received FMT as part of a clinical trial. We found comparable MAIT cell frequencies in healthy controls and mRCC patients. In contrast, γδ T cells exhibited a numerical decline in mRCC, which was partially reversed by FMT. We also found a significant increase in the PB CD4+ MAIT cell compartment of mRCC patients with or without FMT. Paired sample analyses revealed CD69 upregulation on MAIT cells accompanied by decreased PD-1 levels post-FMT. These changes were unique to MAIT cells as non-MAIT T lymphocytes showed either no trend or a trend in the opposite direction. Importantly, FMT did not render MAIT cells exhausted as also judged by their stable expression of TIM-3, LAG-3, BTLA, CTLA-4, TIGIT and VISTA. These findings were corroborated in functional assays in which MAIT cells were stimulated with MR1 ligands or with a combination of IL-12 and IL-18 to produce inflammatory cytokines and granzyme B. Indeed, when stimulated ex vivo with IL-12 and IL-18, MAIT cells mounted a more rigorous TNF-α response post-FMT. In conclusion, FMT improves MAIT cell functions, which should serve patients well in subsequent microbial challenges in the face of cancer-elicited immunosuppression. Trial Registration: https://clinicaltrials.gov/ Identifier: NCT04163289 (registration date: November 14, 2019).

Inducing mismatch repair deficiency sensitizes immune-cold neuroblastoma to anti-CTLA4 and generates broad anti-tumor immune memory.

Immune checkpoint blockade can induce potent and durable responses in patients with highly immunogenic mismatch repair-deficient tumors; however, these drugs are ineffective against immune-cold neuroblastoma tumors. To establish a role for a T cell-based therapy against neuroblastoma, we show that T cell and memory T cell-dependent gene expression are associated with improved survival in high-risk neuroblastoma patients. To stimulate anti-tumor immunity and reproduce this immune phenotype in neuroblastoma tumors, we used CRISPR-Cas9 to knockout MLH1-a crucial molecule in the DNA mismatch repair pathway-to induce mismatch repair deficiency in a poorly immunogenic murine neuroblastoma model. Induced mismatch repair deficiency increased the expression of proinflammatory genes and stimulated T cell infiltration into neuroblastoma tumors. In contrast to adult cancers with induced mismatch repair deficiency, neuroblastoma tumors remained unresponsive to anti-PD1 treatment. However, anti-CTLA4 therapy was highly effective against these tumors. Anti-CTLA4 therapy promoted immune memory and T cell epitope spreading in cured animals. Mechanistically, the effect of anti-CTLA4 therapy against neuroblastoma tumors with induced mismatch repair deficiency is CD4+ T cell dependent, as depletion of these cells abolished the effect. Therefore, a therapeutic strategy involving mismatch repair deficiency-based T cell infiltration of neuroblastoma tumors combined with anti-CTLA4 can serve as a novel T cell-based treatment strategy for neuroblastoma.

The role of gut microbiome in immune modulation in metastatic renal cell carcinoma.

Treatment of metastatic renal cell carcinomas (mRCC) has drastically improved since the advent of immunotherapy with immune checkpoint inhibitors (ICIs), with a significant proportion of patients achieving durable responses. While this has revolutionized treatment and improved outcomes for mRCC patients, a large subset of patients still does not respond to treatment with ICIs. Moreover, ICIs can induce various immune-related adverse events, limiting their use in many patients. Therefore, there is a need to identify the predictive biomarkers of both efficacy and toxicity associated with ICIs, which would allow for a more personalized approach and help with clinical decision-making. This review aims to explore the role of the gut microbiome in RCC to overcome primary resistance and predict response to treatment with ICIs. First, current therapeutic strategies and mechanisms of action of ICI therapies for RCC treatment will be reviewed. With the technological development of shotgun whole-genome sequencing, the gut microbiome has emerged as an exciting field of research within oncology. Thus, the role of the microbiome and its bidirectional interaction with ICIs and other drugs will be explored, with a particular focus on the microbiome profile in RCC. Lastly, the rationale for future clinical interventions to overcome resistance to ICIs using fecal microbiota transplantation in patients with RCC will be presented.

HPV-Positive and -Negative Cervical Cancers Are Immunologically Distinct.

Although infection with human papillomavirus (HPV) is associated with nearly all cervical cancers (CC), a small proportion are HPV-negative. Recently, it has become clear that HPV-negative CC represent a distinct disease phenotype compared to HPV-positive disease and exhibit increased mortality. In addition, variations between different HPV types associated with CC have been linked to altered molecular pathology and prognosis. We compared the immune microenvironments of CC caused by HPV α9 species (HPV16-like), HPV α7 species (HPV18-like) and HPV-negative disease. HPV-negative CC appeared distinct from other subtypes, with greatly reduced levels of lymphocyte infiltration compared to either HPV α9 or α7 CC. Besides reduced levels of markers indicative of B, T, and NK lymphocytes, the expression of T-cell effector molecules, activation/exhaustion markers, and T-cell receptor diversity were also significantly lower in HPV-negative CC. Interestingly, HPV-negative CC expressed much higher levels of potential neoantigens than HPV-positive CC. These results identify profound differences between the immune landscape of HPV-positive and HPV-negative CC as well as modest differences between HPV α9 and α7 CC. These differences may contribute to altered patient outcomes between HPV-negative and HPV-positive CC and potentially between CC associated with different HPV types.

Next Wave of Targets in the Treatment of Advanced Renal Cell Carcinoma.

While surgical resection has remained the mainstay of treatment in early-stage renal cell carcinoma (RCC), therapeutic options in the advanced setting have remarkably expanded over the last 20 years. Tyrosine kinase inhibitors targeting the vascular endothelial growth factor receptor (VEGF-TKIs) and anti-programmed cell death 1 (PD-1)/anti-programmed death-ligand 1 (PD-L1)-based immune checkpoint inhibitors (ICIs) have become globally accepted options in the upfront metastatic setting, with different ICI-based combination strategies improving overall survival compared to single-agent Sunitinib. Although some patients benefit from long-term responses, most eventually develop disease progression. Ongoing efforts to better understand the biology of RCC and the different mechanisms of acquired resistance have led to the identification of promising therapeutic targets. Belzutifan, a novel agent targeting the angiogenic pathway involving hypoxia-inducible factors (HIFs), has already been approved for the treatment of early-stage tumors associated with VHL disease and represents a very promising therapy in advanced RCC. Other putative targets include epigenetic regulation enzymes, as well as several metabolites such as adenosine, glutaminase and tryptophan, which are critical players in cancer cell metabolism and in the tumor microenvironment. Different methods of immune regulation are also being investigated, including CAR-T cell therapy and modulation of the gut microbiome, in addition to novel agents targeting the interleukin-2 (IL-2) pathway. This review aims to highlight the emergent novel therapies for RCC and their respective completed and ongoing clinical trials.

The Effect of the Gut Microbiota on Systemic and Anti-Tumor Immunity and Response to Systemic Therapy against Cancer.

Gut microbiota can have opposing functions from pro-tumorigenic to anti-tumorigenic effects. Increasing preclinical and clinical evidence suggests that the intestinal microbiota affects cancer patients' response to immune checkpoint inhibitors (ICIs) immunotherapy, such as anti-programmed cell death protein 1 (PD-1) and its ligand (PD-L1) and anti-cytotoxic T lymphocyte-associated protein 4 (CTLA-4). Microbiota-induced inflammation possibly contributes to tumor growth and cancer development. Microbiota-derived metabolites can also be converted to carcinogenic agents related to genetic mutations and DNA damage in organs such as the colon. However, other attributes of microbiota, such as greater diversity and specific bacterial species and their metabolites, are linked to better clinical outcomes and potentially improved anti-tumor immunity. In addition, the intratumoral microbial composition strongly affects T-cell-mediated cytotoxicity and anti-tumor immune surveillance, adding more complexity to the cancer-microbiome-immune axis. Despite the emerging clinical evidence for the activity of the gut microbiota in immuno-oncology, the fundamental mechanisms of such activity are not well understood. This review provides an overview of underlying mechanisms by which the gut microbiota and its metabolites enhance or suppress anti-tumor immune responses. Understanding such mechanisms allows for better design of microbiome-specific treatment strategies to improve the clinical outcome in cancer patients undergoing systemic therapy.

Emerging Biomarker-Guided Therapies in Prostate Cancer.

Prostate cancer remains one of the leading causes of cancer death in men worldwide. In the past decade, several new treatments for advanced prostate cancer have been approved. With a wide variety of available drugs, including cytotoxic agents, androgen receptor axis-targeted therapies, and alpha-emitting radiation therapy, identifying their optimal sequencing remains a challenge. Progress in the understanding of the biology of prostate cancer has provided an opportunity for a more refined and personalized treatment selection process. With the advancement of molecular sequencing techniques, genomic precision through the identification of potential treatment targets and predictive biomarkers has been rapidly evolving. In this review, we discussed biomarker-driven treatments for advanced prostate cancer. First, we presented predictive biomarkers for established, global standard treatments for advanced diseases, such as chemotherapy and androgen receptor axis-targeted agents. We also discussed targeted agents with recent approval for special populations, such as poly ADP ribose polymerase (PARP) inhibitors in patients with metastatic castrate-resistant prostate cancer with homologous recombination repair-deficient tumors, pembrolizumab in patients with high levels of microsatellite instability or high tumor mutational burden, and prostate-specific membrane antigen (PSMA) directed radioligand theragnostic treatment for PSMA expressing tumors. Additionally, we discussed evolving treatments, such as cancer vaccines, chimeric antigen receptor T-cells (CAR-T), Bispecific T-cell engagers (BiTEs), other targeted agents such as AKT inhibitors, and various combination treatments. In summary, advances in molecular genetics have begun to propel personalized medicine forward in the management of advanced prostate cancer, allowing for a more precise, biomarker-driven treatment selection with the goal of improving overall efficacy.

Glial activation positron emission tomography imaging in radiation treatment of breast cancer brain metastases.

Brain metastases affect more breast cancer patients than ever before due to increased overall patient survival with improved molecularly targeted treatments. Approximately 25-34% of breast cancer patients develop brain metastases in their lifetime. Due to the blood-brain barrier (BBB), the standard treatment for breast cancer brain metastases (BCBM) is surgery, stereotactic radiosurgery (SRS) and/or whole brain radiation therapy (WBRT). At the cost of cognitive side effects, WBRT has proven efficacy in treating brain metastases when used with local therapies such as SRS and surgery. This review investigated the potential use of glial activation positron emission tomography (PET) imaging for radiation treatment of BCBM. In order to put these studies into context, we provided background on current radiation treatment approaches for BCBM, our current understanding of the brain microenvironment, its interaction with the peripheral immune system, and alterations in the brain microenvironment by BCBM and radiation. We summarized preclinical literature on the interactions between glial activation and cognition and clinical studies using translocator protein (TSPO) PET to image glial activation in the context of neurological diseases. TSPO-PET is not employed clinically in assessing and guiding cancer therapies. However, it has gained traction in preclinical studies where glial activation was investigated from primary brain cancer, metastases and radiation treatments. Novel glial activation PET imaging and its applications in preclinical studies using breast cancer models and glial immunohistochemistry are highlighted. Lastly, we discuss the potential clinical application of glial activation imaging to improve the therapeutic ratio of radiation treatments for BCBM.

Addressing the Elephant in the Immunotherapy Room: Effector T-Cell Priming versus Depletion of Regulatory T-Cells by Anti-CTLA-4 Therapy.

Cytotoxic T-lymphocyte Associated Protein 4 (CTLA-4) is an immune checkpoint molecule highly expressed on regulatory T-cells (Tregs) that can inhibit the activation of effector T-cells. Anti-CTLA-4 therapy can confer long-lasting clinical benefits in cancer patients as a single agent or in combination with other immunotherapy agents. However, patient response rates to anti-CTLA-4 are relatively low, and a high percentage of patients experience severe immune-related adverse events. Clinical use of anti-CTLA-4 has regained interest in recent years; however, the mechanism(s) of anti-CTLA-4 is not well understood. Although activating T-cells is regarded as the primary anti-tumor mechanism of anti-CTLA-4 therapies, mounting evidence in the literature suggests targeting intra-tumoral Tregs as the primary mechanism of action of these agents. Tregs in the tumor microenvironment can suppress the host anti-tumor immune responses through several cell contact-dependent and -independent mechanisms. Anti-CTLA-4 therapy can enhance the priming of T-cells by blockading CD80/86-CTLA-4 interactions or depleting Tregs through antibody-dependent cellular cytotoxicity and phagocytosis. This review will discuss proposed fundamental mechanisms of anti-CTLA-4 therapy, novel uses of anti-CTLA-4 in cancer treatment and approaches to improve the therapeutic efficacy of anti-CTLA-4.

The role of gut microbiome in modulating response to immune checkpoint inhibitor therapy in cancer.

Immunotherapy has led to a paradigm shift in the treatment of several cancers. There have been significant efforts to identify biomarkers that can predict response and toxicities related to immune checkpoint inhibitor (ICPI) therapy. Despite these advances, it has been challenging to tease out why a subset of patients benefit more than others or why certain patients experience immune-related adverse events (irAEs). Although the immune-modulating properties of the human gut bacterial ecosystem are yet to be fully elucidated, there has been growing interest in evaluating the role of the gut microbiome in shaping the therapeutic response to cancer immunotherapy. Considerable research efforts are currently directed to utilizing metagenomic and metabolic profiling of stool microbiota in patients on ICPI-based therapies. Dysbiosis or loss of microbial diversity has been associated with a poor treatment response to ICPIs and worse survival outcomes in cancer patients. Emerging data have shown that certain bacterial strains, such as Faecalibacterium that confer sensitivity to ICPI, also have a higher propensity to increase the risk of irAEs. Additionally, the microbiome can modulate the local immune response at the intestinal interface and influence the trafficking of bacterial peptide primed T-cells distally, influencing the toxicity patterns to ICPI. Antibiotic or diet induced alterations in composition of the microbiome can also indirectly alter the production of certain bacterial metabolites such as deoxycholate and short chain fatty acids that can influence the anti-tumor tolerogenesis. Gaining sufficient understanding of the exact mechanisms underpinning the interplay between ICPI induced anti-tumor immunity and the immune modulatory role gut microbiome can be vital in identifying potential avenues of improving outcomes to cancer immunotherapy. In the current review, we have summarized and highlighted the key emerging data supporting the role of gut microbiome in regulating response to ICPIs in cancer.

Immunosuppressive Effects of Myeloid-Derived Suppressor Cells in Cancer and Immunotherapy.

The primary function of myeloid cells is to protect the host from infections. However, during cancer progression or states of chronic inflammation, these cells develop into myeloid-derived suppressor cells (MDSCs) that play a prominent role in suppressing anti-tumor immunity. Overcoming the suppressive effects of MDSCs is a major hurdle in cancer immunotherapy. Therefore, understanding the mechanisms by which MDSCs promote tumor growth is essential for improving current immunotherapies and developing new ones. This review explores mechanisms by which MDSCs suppress T-cell immunity and how this impacts the efficacy of commonly used immunotherapies.