Identification of bacteria-derived HLA-bound peptides in melanoma.

A variety of species of bacteria are known to colonize human tumours1-11, proliferate within them and modulate immune function, which ultimately affects the survival of patients with cancer and their responses to treatment12-14. However, it is not known whether antigens derived from intracellular bacteria are presented by the human leukocyte antigen class I and II (HLA-I and HLA-II, respectively) molecules of tumour cells, or whether such antigens elicit a tumour-infiltrating T cell immune response. Here we used 16S rRNA gene sequencing and HLA peptidomics to identify a peptide repertoire derived from intracellular bacteria that was presented on HLA-I and HLA-II molecules in melanoma tumours. Our analysis of 17 melanoma metastases (derived from 9 patients) revealed 248 and 35 unique HLA-I and HLA-II peptides, respectively, that were derived from 41 species of bacteria. We identified recurrent bacterial peptides in tumours from different patients, as well as in different tumours from the same patient. Our study reveals that peptides derived from intracellular bacteria can be presented by tumour cells and elicit immune reactivity, and thus provides insight into a mechanism by which bacteria influence activation of the immune system and responses to therapy.

EMT activates exocytotic Rabs to coordinate invasion and immunosuppression in lung cancer.

Epithelial-to-mesenchymal transition (EMT) underlies immunosuppression, drug resistance, and metastasis in epithelial malignancies. However, the way in which EMT orchestrates disparate biological processes remains unclear. Here, we identify an EMT-activated vesicular trafficking network that coordinates promigratory focal adhesion dynamics with an immunosuppressive secretory program in lung adenocarcinoma (LUAD). The EMT-activating transcription factor ZEB1 drives exocytotic vesicular trafficking by relieving Rab6A, Rab8A, and guanine nucleotide exchange factors from miR-148a-dependent silencing, thereby facilitating MMP14-dependent focal adhesion turnover in LUAD cells and autotaxin-mediated CD8+ T cell exhaustion, indicating that cell-intrinsic and extrinsic processes are linked through a microRNA that coordinates vesicular trafficking networks. Blockade of ZEB1-dependent secretion reactivates antitumor immunity and negates resistance to PD-L1 immune checkpoint blockade, an important clinical problem in LUAD. Thus, EMT activates exocytotic Rabs to drive a secretory program that promotes invasion and immunosuppression in LUAD.

Cabozantinib monotherapy for advanced adrenocortical carcinoma: a single-arm, phase 2 trial.

BACKGROUND: Adrenocortical carcinoma is a rare malignancy with poor response to systemic chemotherapy. Mitotane is the only approved therapy for adrenocortical carcinoma. Cabozantinib is a multikinase inhibitor approved in multiple malignancies. This is the first prospective trial to explore the anti-tumour activity, safety, and pharmacokinetic profile of cabozantinib in patients with advanced adrenocortical carcinoma. METHODS: This investigator-initiated, single-arm, phase 2 trial in adult patients (aged ≥18 years) with advanced adrenocortical carcinoma was done at the University of Texas MD Anderson Cancer Center (Houston, TX, USA). Eligible patients had histologically confirmed adrenocortical carcinoma, were not candidates for surgery with curative intent, had measurable disease, had an estimated life expectancy of at least 3 months, and an Eastern Cooperative Oncology Group (ECOG) performance status of 0-2 with adequate organ function. Patients who had used mitotane within 6 months of study participation were required to have a serum mitotane level of less than 2 mg/L. Patients were given oral cabozantinib 60 mg daily with the option of dose reduction to manage adverse events. The primary endpoint was progression-free survival at 4 months, assessed in all patients who received at least one dose of study drug per protocol. This study is registered with ClinicalTrials.gov, NCT03370718, and is now complete. FINDINGS: Between March 1, 2018, and May 31, 2021, we enrolled 18 patients (ten males and eight females), all of whom received at least one dose of study treatment. Of the 18 patients, eight (44%) had an ECOG performance status of 0, nine (50%) patients had a performance status of 1, and one (6%) patient had a performance status of 2. Median follow-up was 36·8 months (IQR 30·2-50·3). At 4 months, 13 (72·2%; 95% CI 46·5-90·3) of 18 patients had progression-free survival and median progression-free survival was 6 months (95% CI 4·3 to not reached). One patient remains on treatment. Treatment-related adverse events of grade 3 or worse occurred in 11 (61%) of 18 patients. The most common grade 3 adverse events were lipase elevation (three [17%] of 18 patients), elevated γ-glutamyl transferase concentrations (two [11%] patients), elevated alanine aminotransferase concentrations (two [11%] patients), hypophosphatemia (two [11%] patients), and hypertension (two [11%] patients). One (6%) of 18 patients had grade 4 hypertension. No treatment related deaths occurred on study. INTERPRETATION: Cabozantinib in advanced adrenocortical carcinoma showed promising efficacy with a manageable and anticipated safety profile. Further prospective studies with cabozantinib alone and in combination with immune checkpoint therapy are ongoing. FUNDING: Exelixis.

Loss of Rnf43 Accelerates Kras-Mediated Neoplasia and Remodels the Tumor Immune Microenvironment in Pancreatic Adenocarcinoma.

BACKGROUND & AIMS: RNF43 is an E3 ubiquitin ligase that is recurrently mutated in pancreatic ductal adenocarcinoma (PDAC) and precursor cystic neoplasms of the pancreas. The impact of RNF43 mutations on PDAC is poorly understood and autochthonous models have not been characterized sufficiently. In this study, we describe a genetically engineered mouse model (GEMM) of PDAC with conditional expression of oncogenic Kras and deletion of the catalytic domain of Rnf43 in exocrine cells. METHODS: We generated Ptf1a-Cre;LSL-KrasG12D;Rnf43flox/flox (KRC) and Ptf1a-Cre; LSL-KrasG12D (KC) mice and animal survival was assessed. KRC mice were sacrificed at 2 months, 4 months, and at moribund status followed by analysis of pancreata by single-cell RNA sequencing. Comparative analyses between moribund KRC and a moribund Kras/Tp53-driven PDAC GEMM (KPC) was performed. Cell lines were isolated from KRC and KC tumors and interrogated by cytokine array analyses, ATAC sequencing, and in vitro drug assays. KRC GEMMs were also treated with an anti-CTLA4 neutralizing antibody with treatment response measured by magnetic response imaging. RESULTS: We demonstrate that KRC mice display a marked increase in incidence of high-grade cystic lesions of the pancreas and PDAC compared with KC. Importantly, KRC mice have a significantly decreased survival compared with KC mice. Using single-cell RNA sequencing, we demonstrated that KRC tumor progression is accompanied by a decrease in macrophages, as well as an increase in T and B lymphocytes, with evidence of increased immune checkpoint molecule expression and affinity maturation, respectively. This was in stark contrast to the tumor immune microenvironment observed in the KPC PDAC GEMM. Furthermore, expression of the chemokine CXCL5 was found to be specifically decreased in KRC cancer cells by means of epigenetic regulation and emerged as a putative candidate for mediating the unique KRC immune landscape. CONCLUSIONS: The KRC GEMM establishes RNF43 as a bona fide tumor suppressor gene in PDAC. This GEMM features a markedly different immune microenvironment compared with previously reported PDAC GEMMs and puts forth a rationale for an immunotherapy approach in this subset of PDAC cases.

Differential Effects of Pancreatic Cancer-Derived Extracellular Vesicles Driving a Suppressive Environment.

Pancreatic ductal adenocarcinoma (PDAC) cells display extensive crosstalk with their surrounding environment to regulate tumor growth, immune evasion, and metastasis. Recent advances have attributed many of these interactions to intercellular communication mediated by small extracellular vesicles (sEVs), involving cancer-associated fibroblasts (CAF). To explore the impact of sEVs on monocyte lineage transition as well as the expression of checkpoint receptors and activation markers, peripheral blood monocytes from healthy subjects were exposed to PDAC-derived sEVs. Additionally, to analyze the role of sEV-associated HA in immune regulation and tissue-resident fibroblasts, monocytes and pancreatic stellate cells were cultured in the presence of PDAC sEVs with or depleted of HA. Exposure of monocytes to sEVs resulted in unique phenotypic changes in HLA-DR, PD-L1, CD86 and CD64 expression, and cytokine secretion that was HA-independent except for IL-1ß and MIP1ß. In contrast, monocyte suppression of autologous T cell proliferation was reduced following exposure to HA-low sEVs. In addition, exposure of stellate cells to sEVs upregulated the secretion of various cytokines, including MMP-9, while removal of HA from PDAC-derived sEVs attenuated the secretion of MMP-9, demonstrating the role of sEV-associated HA in regulating expression of this pro-tumorigenic cytokine from stellate cells. This observation lends credence to the findings from the TCGA database that PDAC patients with high levels of enzymes in the HA synthesis pathway had worse survival rates compared with patients having low expression of these enzymes. PDAC-derived sEVs have an immune modulatory role affecting the activation state of monocyte subtypes. However, sEV-associated HA does not affect monocyte phenotype but alters cytokine secretion and suppression of autologous T cell proliferation and induces secretion of pro-tumorigenic factors by pancreatic stellate cells (PSC), as has been seen following the conversion of PSCs to cancer-associated fibroblasts (CAFs). Interruption of the hexosamine biosynthetic pathway, activated in PDAC producing the key substrate (UDP-GlcNAc) for HA synthesis, thus, represents a potential clinical interception strategy for PDAC patients. Findings warrant further investigations of underlying mechanisms involving larger sample cohorts.

Durvalumab plus tremelimumab in advanced or metastatic soft tissue and bone sarcomas: a single-centre phase 2 trial.

BACKGROUND: Few standard treatment options are available for patients with metastatic sarcomas. We did this trial to evaluate the efficacy, safety, and changes in the tumour microenvironment for durvalumab, an anti-PD-L1 drug, and tremelimumab, an anti-CTLA-4 drug, across multiple sarcoma subtypes. METHODS: In this single-centre phase 2 trial, done at The University of Texas MD Anderson Cancer Center (Houston, TX USA), patients aged 18 years or older with advanced or metastatic sarcoma with an Eastern Cooperative Oncology Group performance status of 0 or 1 who had received at least one previous line of systemic therapy were enrolled in disease subtype-specific groups (liposarcoma, leiomyosarcoma, angiosarcoma, undifferentiated pleomorphic sarcoma, synovial sarcoma, osteosarcoma, alveolar soft-part sarcoma, chordoma, and other sarcomas). Patients received 1500 mg intravenous durvalumab and 75 mg intravenous tremelimumab for four cycles, followed by durvalumab alone every 4 weeks for up to 12 months. The primary endpoint was progression-free survival at 12 weeks in the intention-to-treat population (all patients who received at least one dose of treatment). Safety was also analysed in the intention-to-treat population. This trial is registered with ClinicalTrials.gov, NCT02815995, and is completed. FINDINGS: Between Aug 17, 2016, and April 9, 2018, 62 patients were enrolled, of whom 57 (92%) received treatment and were included in the intention-to-treat population. With a median follow-up of 37·2 months (IQR 1·8-10·1), progression-free survival at 12 weeks was 49% (95% CI 36-61). 21 grade 3-4 treatment-related adverse events were reported, the most common of which were increased lipase (four [7%] of 57 patients), colitis (three [5%] patients), and pneumonitis (three [5%] patients). Nine (16%) patients had a treatment related serious adverse event. One patient had grade 5 pneumonitis and colitis. INTERPRETATION: The combination of durvalumab and tremelimumab is an active treatment regimen for advanced or metastatic sarcoma and merits evaluation in specific subsets in future trials. FUNDING: AstraZeneca.

Effector memory cytotoxic CD3+/CD8+/CD45RO+ T cells are predictive of good survival and a lower risk of recurrence in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) with high tumour-infiltrating lymphocytes (TILs) has been associated with a promising prognosis. To better understand the prognostic value of immune cell subtypes in TNBC, we characterised TILs and the interaction between tumour cells and immune cell subtypes. A total of 145 breast cancer tissues were stained by multiplex immunofluorescence (mIF), including panel 1 (PD-L1, PD-1, CD3, CD8, CD68 and CK) and panel 2 (Foxp3, Granzyme B, CD45RO, CD3, CD8 and CK). Phenotypes were analysed and quantified by pathologists using InForm software. We found that in the ER-negative (ER <1% and HER2-negative) group and the ER/PR-low positive (ER 1-9% and HER2-negative) group, 11.2% and 7.1% of patients were PD-L1+ by the tumour cell score, 29.0% and 28.6% were PD-L1+ by the modified immune cell score and 30.8% and 32.1% were PD-L1+ by the combined positive score. We combined ER-negative and ER/PR-low positive cases for the survival analysis since a 10% cut-off is often used in clinical practice for therapeutic purposes. The densities of PD-L1+ tumour cells (HR: 0.366, 95% CI: 0.138-0.970; p = 0.043) within the tumour compartment and CD3+ immune cells in the total area (tumour and stromal compartments combined) (HR: 0.213, 95% CI: 0.070-0.642; p = 0.006) were favourable prognostic biomarkers for overall survival (OS) in TNBC. The density of effector/memory cytotoxic T cells (CD3+CD8+CD45RO+) in the tumour compartment was an independent prognostic biomarker for OS (HR: 0.232, 95% CI: 0.086-0.628; p = 0.004) and DFS (HR: 0.183, 95% CI: 0.1301-0.744; p = 0.009) in TNBC. Interestingly, spatial data suggested that patients with a higher density of PD-L1+ tumour cells had shorter cell-cell distances from tumour cells to cytotoxic T cells (p < 0.01). In conclusion, we found that phenotyping tumour immune cells by mIF is highly informative in understanding the immune microenvironment in TNBC. PD-L1+ tumour cells, total T cells and effector/memory cytotoxic T cells are promising prognostic biomarkers in TNBC.

2022 Practice Recommendation Updates From the World Consensus Conference on BIA-ALCL.

BACKGROUND: Laboratory and clinical research on breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is rapidly evolving. Changes in standard of care and insights into best practice were recently presented at the 3rd World Consensus Conference on BIA-ALCL. OBJECTIVES: The authors sought to provide practice recommendations from a consensus of experts, supplemented with a literature review regarding epidemiology, etiology, pathogenesis, diagnosis, treatment, socio-psychological aspects, and international authority guidance. METHODS: A literature search of all manuscripts between 1997 and August 2021 for the above areas of BIA-ALCL was conducted with the PubMed database. Manuscripts in different languages, on non-human subjects, and/or discussing conditions separate from BIA-ALCL were excluded. The study was conducted employing the Delphi process, gathering 18 experts panelists and utilizing email-based questionnaires to record the level of agreement with each statement by applying a 5-point Likert Scale. Median response, interquartile range, and comments were employed to accept, reject, or revise each statement. RESULTS: The literature search initially yielded 764 manuscripts, of which 405 were discarded. From the remaining 359, only 218 were included in the review and utilized to prepare 36 statements subdivided into 5 sections. After 1 round, panelists agreed on all criteria. CONCLUSIONS: BIA-ALCL is uncommon and still largely underreported. Mandatory implant registries and actions by regulatory authorities are needed to better understand disease epidemiology and address initial lymphomagenesis and progression. Deviation from current diagnosis and treatment protocols can lead to disease recurrence, and research on breast implant risk factors provide insight to etiology.

Aurora kinase inhibition sensitizes melanoma cells to T-cell-mediated cytotoxicity.

Although immunotherapy has achieved impressive durable clinical responses, many cancers respond only temporarily or not at all to immunotherapy. To find novel, targetable mechanisms of resistance to immunotherapy, patient-derived melanoma cell lines were transduced with 576 open reading frames, or exposed to arrayed libraries of 850 bioactive compounds, prior to co-culture with autologous tumor-infiltrating lymphocytes (TILs). The synergy between the targets and TILs to induce apoptosis, and the mechanisms of inhibiting resistance to TILs were interrogated. Gene expression analyses were performed on tumor samples from patients undergoing immunotherapy for metastatic melanoma. Finally, the effect of inhibiting the top targets on the efficacy of immunotherapy was investigated in multiple preclinical models. Aurora kinase was identified as a mediator of melanoma cell resistance to T-cell-mediated cytotoxicity in both complementary screens. Aurora kinase inhibitors were validated to synergize with T-cell-mediated cytotoxicity in vitro. The Aurora kinase inhibition-mediated sensitivity to T-cell cytotoxicity was shown to be partially driven by p21-mediated induction of cellular senescence. The expression levels of Aurora kinase and related proteins were inversely correlated with immune infiltration, response to immunotherapy and survival in melanoma patients. Aurora kinase inhibition showed variable responses in combination with immunotherapy in vivo, suggesting its activity is modified by other factors in the tumor microenvironment. These data suggest that Aurora kinase inhibition enhances T-cell cytotoxicity in vitro and can potentiate antitumor immunity in vivo in some but not all settings. Further studies are required to determine the mechanism of primary resistance to this therapeutic intervention.

Pulmonary resection for tissue harvest in adoptive tumor-infiltrating lymphocyte therapy: Safety and feasibility.

BACKGROUND AND OBJECTIVES: Adoptive T-cell therapies (ACTs) using expansion of tumor-infiltrating lymphocyte (TIL) populations are of great interest for advanced malignancies, with promising response rates in trial settings. However, postoperative outcomes following pulmonary TIL harvest have not been widely documented, and surgeons may be hesitant to operate in the setting of widespread disease. METHODS: Patients who underwent pulmonary TIL harvest were identified, and postoperative outcomes were studied, including pulmonary, cardiovascular, infectious, and wound complications. RESULTS: 83 patients met inclusion criteria. Pulmonary TIL harvest was undertaken primarily via a thoracoscopy with a median operative blood loss and duration of 30 ml and 65 min, respectively. The median length of stay was 2 days. Postoperative events were rare, occurring in only five (6%) patients, including two discharged with a chest tube, one discharged with oxygen, one episode of urinary retention, and one blood transfusion. No reoperations occurred. The median time from TIL harvest to ACT infusion was 37 days. CONCLUSIONS: Pulmonary TIL harvest is safe and feasible, without major postoperative events in our cohort. All patients were able to receive intended ACT infusion without delays. Therefore, thoracic surgeons should actively participate in ongoing ACT trials and aggressively seek to enroll patients on these protocols.

Exposure to anti-PD-1 causes functional differences in tumor-infiltrating lymphocytes in rare solid tumors.

The pervasive use of therapeutic antibodies targeting programmed cell death protein 1 (PD-1) to boost anti-tumor immunity has positioned this approach to become the standard-of-care for some solid tumor malignancies. However, little is known as to how blockade of PD-1 may alter the function or phenotype of tumor-infiltrating lymphocytes (TIL). We used our ongoing Phase II clinical trial of pembrolizumab for patients with rare solid tumors from various types (NCT02721732) with matched core biopsies taken at baseline and after initial dose of anti-PD-1 (15-21 days post-dose) to elucidate this question. One fresh core needle biopsy was used to propagate TIL ex vivo to analyze phenotype and function using flow cytometry in both CD8+ and CD4+ TIL populations. An enriched CTLA-4 expression in the CD4+ TIL population was observed in TIL expanded from the on-treatment samples compared to TIL expanded from the matched baseline (n = 22, p = 0.0007) but was not observed in patients who experienced tumor regression. Impact on functionality was evaluated by measuring secretion of 65 soluble factors by expanded TIL from 26 patients at baseline and on-treatment. The CD8+ TIL population demonstrated a diminished cytokine secretion profile post-pembrolizumab. Overall, our study assesses the ramifications of one dose of anti-PD-1 on TIL in rare solid tumor types.

Potential clinical application of tumor-infiltrating lymphocyte therapy for ovarian epithelial cancer prior or post-resistance to chemotherapy.

BACKGROUND: Immunotherapy has become a powerful treatment option for several solid tumor types. The presence of tumor-infiltrating lymphocytes (TIL) is correlated with better prognosis in ovarian cancer, pointing at the possibility to benefit from harnessing their anti-tumor activity. This preclinical study explores the feasibility of adoptive cell therapy (ACT) with TIL using an improved culture method. METHODS: TIL from high-grade serous ovarian cancer were cultured using a combination of IL-2 with agonistic antibodies targeting 4-1BB and CD3. The cells were phenotyped using flow cytometry in the fresh tissue and after expansion. Tumor reactivity was assessed against HLA-matched ovarian cancer cell lines via IFN-γ ELISPOT. RESULTS: Ovarian cancer is highly infiltrated with CD8+ TIL that are preferentially and robustly expanded with the addition of the agonistic antibodies. With a 95% success rate, the TIL are grown to ≥ 100 × 106 cells in 2-3 weeks without over differentiation. In addition, the CD8+ TIL grown with this method showed HLA-restricted tumor recognition. CONCLUSIONS: These results indicate the viability of TIL ACT for refractory ovarian cancer by allowing for the large expansion of anti-tumor TIL in a short time and consistent manner.

Novel algorithmic approach predicts tumor mutation load and correlates with immunotherapy clinical outcomes using a defined gene mutation set.

BACKGROUND: While clinical outcomes following immunotherapy have shown an association with tumor mutation load using whole exome sequencing (WES), its clinical applicability is currently limited by cost and bioinformatics requirements. METHODS: We developed a method to accurately derive the predicted total mutation load (PTML) within individual tumors from a small set of genes that can be used in clinical next generation sequencing (NGS) panels. PTML was derived from the actual total mutation load (ATML) of 575 distinct melanoma and lung cancer samples and validated using independent melanoma (n = 312) and lung cancer (n = 217) cohorts. The correlation of PTML status with clinical outcome, following distinct immunotherapies, was assessed using the Kaplan-Meier method. RESULTS: PTML (derived from 170 genes) was highly correlated with ATML in cutaneous melanoma and lung adenocarcinoma validation cohorts (R2 = 0.73 and R2 = 0.82, respectively). PTML was strongly associated with clinical outcome to ipilimumab (anti-CTLA-4, three cohorts) and adoptive T-cell therapy (1 cohort) clinical outcome in melanoma. Clinical benefit from pembrolizumab (anti-PD-1) in lung cancer was also shown to significantly correlate with PTML status (log rank P value < 0.05 in all cohorts). CONCLUSIONS: The approach of using small NGS gene panels, already applied to guide employment of targeted therapies, may have utility in the personalized use of immunotherapy in cancer.

Antigen-specific effector CD4 T lymphocytes school lamina propria dendritic cells to transfer innate tolerance.

Dendritic cells (DCs) have been shown to play a major role in oral tolerance, and this function has been associated with their ability to produce anti-inflammatory cytokines and to induce suppressive regulatory T cells. In this study, we demonstrate that upon oral administration of Ag, lamina propia (LP) DCs engage specific T cells and acquire a novel mechanism by which they transfer tolerance against diverse T cell specificities. Indeed, when Ig-myelin oligodendrocyte glycoprotein (MOG) carrying the MOG(35-55) epitope was orally administered into either T cell-sufficient or -deficient mice, only the T cell-sufficient hosts yielded CD8α(+) and CD8α(-) LP DCs that were able to transfer tolerance to a variety of MHC class II-restricted effector T cells. Surprisingly, these LP DCs upregulated programmed cell death ligand 1 during the initial interaction with MOG-specific T cells and used this inhibitory molecule to suppress activation of T cells regardless of Ag specificity. Furthermore, oral Ig-MOG was able to overcome experimental autoimmune encephalomyelitis induced with CNS homogenate, indicating that the DCs are able to modulate disease involving diverse T cell specificities. This previously unrecognized attribute potentiates DCs against autoimmunity.

Bone marrow-derived IL-13Rα1-positive thymic progenitors are restricted to the myeloid lineage.

The earliest thymic progenitors (ETPs) were recently shown to give rise to both lymphoid and myeloid cells. Whereas the majority of ETPs are derived from IL-7Rα-positive cells and give rise exclusively to T cells, the origin of the myeloid cells remains undefined. In this study, we show both in vitro and in vivo that IL-13Rα1(+) ETPs yield myeloid cells with no potential for maturation into T cells, whereas IL-13Rα1(-) ETPs lack myeloid potential. Moreover, transfer of lineage-negative IL-13Rα1(+) bone marrow stem cells into IL-13Rα1-deficient mice reconstituted thymic IL-13Rα1(+) myeloid ETPs. Myeloid cells or macrophages in the thymus are regarded as phagocytic cells whose function is to clear apoptotic debris generated during T cell development. However, the myeloid cells derived from IL-13Rα1(+) ETPs were found to perform Ag-presenting functions. Thus, IL-13Rα1 defines a new class of myeloid restricted ETPs yielding APCs that could contribute to development of T cells and the control of immunity and autoimmunity.

Interpretable Spatial Gradient Analysis for Spatial Transcriptomics Data.

Cellular anatomy and signaling vary across niches, which can induce gradated gene expressions in subpopulations of cells. Such spatial transcriptomic gradient (STG) makes a significant source of intratumor heterogeneity and can influence tumor invasion, progression, and response to treatment. Here we report Local Spatial Gradient Inference (LSGI), a computational framework that systematically identifies spatial locations with prominent, interpretable STGs from spatial transcriptomic (ST) data. To achieve so, LSGI scrutinizes each sliding window employing non-negative matrix factorization (NMF) combined with linear regression. With LSGI, we demonstrated the identification of spatially proximal yet opposite directed pathway gradients in a glioblastoma dataset. We further applied LSGI to 87 tumor ST datasets reported from nine published studies and identified both pan-cancer and tumor-type specific pathways with gradated expression patterns, such as epithelial mesenchymal transition, MHC complex, and hypoxia. The local gradients were further categorized according to their association to tumor-TME (tumor microenvironment) interface, highlighting the pathways related to spatial transcriptional intratumoral heterogeneity. We conclude that LSGI enables highly interpretable STG analysis which can reveal novel insights in tumor biology from the increasingly reported tumor ST datasets.

Elucidating immune-related gene transcriptional programs via factorization of large-scale RNA-profiles.

Recent developments in immunotherapy, including immune checkpoint blockade (ICB) and adoptive cell therapy (ACT), have encountered challenges such as immune-related adverse events and resistance, especially in solid tumors. To advance the field, a deeper understanding of the molecular mechanisms behind treatment responses and resistance is essential. However, the lack of functionally characterized immune-related gene sets has limited data-driven immunological research. To address this gap, we adopted non-negative matrix factorization on 83 human bulk RNA sequencing (RNA-seq) datasets and constructed 28 immune-specific gene sets. After rigorous immunologist-led manual annotations and orthogonal validations across immunological contexts and functional omics data, we demonstrated that these gene sets can be applied to refine pan-cancer immune subtypes, improve ICB response prediction and functionally annotate spatial transcriptomic data. These functional gene sets, informing diverse immune states, will advance our understanding of immunology and cancer research.

Elucidating immune-related gene transcriptional programs via factorization of large-scale RNA-profiles.

Recent developments in immunotherapy, including immune checkpoint blockade (ICB) and adoptive cell therapy, have encountered challenges such as immune-related adverse events and resistance, especially in solid tumors. To advance the field, a deeper understanding of the molecular mechanisms behind treatment responses and resistance is essential. However, the lack of functionally characterized immune-related gene sets has limited data-driven immunological research. To address this gap, we adopted non-negative matrix factorization on 83 human bulk RNA-seq datasets and constructed 28 immune-specific gene sets. After rigorous immunologist-led manual annotations and orthogonal validations across immunological contexts and functional omics data, we demonstrated that these gene sets can be applied to refine pan-cancer immune subtypes, improve ICB response prediction and functionally annotate spatial transcriptomic data. These functional gene sets, informing diverse immune states, will advance our understanding of immunology and cancer research.

Identification of Colorectal Cancer Cell Stemness from Single-Cell RNA Sequencing.

Cancer stem cells (CSC) play a critical role in metastasis, relapse, and therapy resistance in colorectal cancer. While characterization of the normal lineage of cell development in the intestine has led to the identification of many genes involved in the induction and maintenance of pluripotency, recent studies suggest significant heterogeneity in CSC populations. Moreover, while many canonical colorectal cancer CSC marker genes have been identified, the ability to use these classical markers to annotate stemness at the single-cell level is limited. In this study, we performed single-cell RNA sequencing on a cohort of 6 primary colon, 9 liver metastatic tumors, and 11 normal (nontumor) controls to identify colorectal CSCs at the single-cell level. Finding poor alignment of the 11 genes most used to identify colorectal CSC, we instead extracted a single-cell stemness signature (SCS_sig) that robustly identified "gold-standard" colorectal CSCs that expressed all marker genes. Using this SCS_sig to quantify stemness, we found that while normal epithelial cells show a bimodal distribution, indicating distinct stem and differentiated states, in tumor epithelial cells stemness is a continuum, suggesting greater plasticity in these cells. The SCS_sig score was quite variable between different tumors, reflective of the known transcriptomic heterogeneity of CRC. Notably, patients with higher SCS_sig scores had significantly shorter disease-free survival time after curative intent surgical resection, suggesting stemness is associated with relapse. IMPLICATIONS: This study reveals significant heterogeneity of expression of genes commonly used to identify colorectal CSCs, and identifies a novel stemness signature to identify these cells from scRNA-seq data.