Two independent exome sequencing initiatives aimed to identify new genes involved in the predisposition to nonpolyposis colorectal cancer led to the identification of heterozygous loss-of-function... Show moreTwo independent exome sequencing initiatives aimed to identify new genes involved in the predisposition to nonpolyposis colorectal cancer led to the identification of heterozygous loss-of-function variants in NPAT, a gene that encodes a cyclin E/CDK2 effector required for S phase entry and a coactivator of histone transcription, in two families with multiple members affected with colorectal cancer. Enrichment of loss-of-function and predicted deleterious NPAT variants was identified in familial/early-onset colorectal cancer patients compared to non-cancer gnomAD individuals, further supporting the association with the disease. Previous studies in Drosophila models showed that NPAT abrogation results in chromosomal instability, increase of double strand breaks, and induction of tumour formation. In line with these results, colorectal cancers with NPAT somatic variants and no DNA repair defects have significantly higher aneuploidy levels than NPAT-wildtype colorectal cancers. In conclusion, our findings suggest that constitutional inactivating NPAT variants predispose to mismatch repair-proficient nonpolyposis colorectal cancer. Show less
Verschoor, Y.L.; Haar, J. van de; Berg, J.G. van den; Sandick, J.W. van; Kodach, L.L.; Dieren, J.M. van; ... ; Chalabi, M. 2024
CD4(+) T cells can "help" or "license" conventional type 1 dendritic cells (cDC1s) to induce CD8(+) cytotoxic T lymphocyte (CTL) anticancer responses, as proven in mouse models. We recently... Show moreCD4(+) T cells can "help" or "license" conventional type 1 dendritic cells (cDC1s) to induce CD8(+) cytotoxic T lymphocyte (CTL) anticancer responses, as proven in mouse models. We recently identified cDC1s with a transcriptomic imprint of CD4(+) T-cell help, specifically in T-cell-infiltrated human cancers, and these cells were associated with a good prognosis and response to PD-1-targeting immunotherapy. Here, we delineate the mechanism of cDC1 licensing by CD4(+) T cells in humans. Activated CD4(+) T cells produce IFN beta via the STING pathway, which promotes MHC-I antigen (cross-)presentation by cDC1s and thereby improves their ability to induce CTL anticancer responses. In cooperation with CD40 ligand (L), IFN beta also optimizes the costimulatory and other functions of cDC1s required for CTL response induction. IFN-I-producing CD4(+) T cells are present in diverse T-cell-infiltrated cancers and likely deliver "help" signals to CTLs locally, according to their transcriptomic profile and colocalization with "helped/licensed" cDCs and tumor-reactive CD8(+) T cells. In agreement with this scenario, the presence of IFN-I-producing CD4(+) T cells in the TME is associated with overall survival and the response to PD-1 checkpoint blockade in cancer patients. Show less
Verschoor, Y.L.; Haar, J. van de; Berg, J.G. van den; Sandick, J.W. van; Kodach, L.L.; Dieren, J.M. van; ... ; Chalabi, M. 2024
Gastric and gastroesophageal junction (G/GEJ) cancers carry a poor prognosis, and despite recent advancements, most patients die of their disease. Although immune checkpoint blockade became part of... Show moreGastric and gastroesophageal junction (G/GEJ) cancers carry a poor prognosis, and despite recent advancements, most patients die of their disease. Although immune checkpoint blockade became part of the standard-of-care for patients with metastatic G/GEJ cancers, its efficacy and impact on the tumor microenvironment (TME) in early disease remain largely unknown. We hypothesized higher efficacy of neoadjuvant immunotherapy plus chemotherapy in patients with nonmetastatic G/GEJ cancer. In the phase 2 PANDA trial, patients with previously untreated resectable G/GEJ tumors (n = 21) received neoadjuvant treatment with one cycle of atezolizumab monotherapy followed by four cycles of atezolizumab plus docetaxel, oxaliplatin and capecitabine. Treatment was well tolerated. There were grade 3 immune-related adverse events in two of 20 patients (10%) but no grade 4 or 5 immune-related adverse events, and all patients underwent resection without treatment-related delays, meeting the primary endpoint of safety and feasibility. Tissue was obtained at multiple time points, allowing analysis of the effects of single-agent anti-programmed cell death ligand 1 (PD-L1) and the subsequent combination with chemotherapy on the TME. Twenty of 21 patients underwent surgery and were evaluable for secondary pathologic response and survival endpoints, and 19 were evaluable for exploratory translational analyses. A major pathologic response (<= 10% residual viable tumor) was observed in 14 of 20 (70%, 95% confidence interval 46-88%) patients, including 9 (45%, 95% confidence interval 23-68%) pathologic complete responses. At a median follow-up of 47 months, 13 of 14 responders were alive and disease-free, and five of six nonresponders had died as a result of recurrence. Notably, baseline anti-programmed cell death protein 1 (PD-1)+CD8+ T cell infiltration was significantly higher in responders versus nonresponders, and comparison of TME alterations following anti-PD-L1 monotherapy versus the subsequent combination with chemotherapy showed an increased immune activation on single-agent PD-1/L1 axis blockade. On the basis of these data, monotherapy anti-PD-L1 before its combination with chemotherapy warrants further exploration and validation in a larger cohort of patients with nonmetastatic G/GEJ cancer. ClinicalTrials.gov registration: NCT03448835.A neoadjuvant treatment regimen of anti-PD-L1 monotherapy followed by anti-PD-L1 plus chemotherapy was well tolerated and led to a major pathologic response rate of 70% in patients with resectable gastric or gastroesophageal junction adenocarcinoma. Show less
Precision oncology approaches for patients with colorectal cancer (CRC) continue to lag behind other solid cancers. Functional precision oncology—a strategy that is based on perturbing primary... Show morePrecision oncology approaches for patients with colorectal cancer (CRC) continue to lag behind other solid cancers. Functional precision oncology—a strategy that is based on perturbing primary tumor cells from cancer patients—could provide a road forward to personalize treatment. We extend this paradigm to measuring proteome activity landscapes by acquiring quantitative phosphoproteomic data from patient-derived organoids (PDOs). We show that kinase inhibitors induce inhibitor- and patient-specific off-target effects and pathway crosstalk. Reconstruction of the kinase networks revealed that the signaling rewiring is modestly affected by mutations. We show non-genetic heterogeneity of the PDOs and upregulation of stemness and differentiation genes by kinase inhibitors. Using imaging mass-cytometry-based profiling of the primary tumors, we characterize the tumor microenvironment (TME) and determine spatial heterocellular crosstalk and tumor-immune cell interactions. Collectively, we provide a framework for inferring tumor cell intrinsic signaling and external signaling from the TME to inform precision (immuno-) oncology in CRC. Show less
Vietsch, E.E.; Latifi, D.; Verheij, M.; Oost, E.W.A. van der; Wilde, R.F. de; Haen, R.; ... ; Eijck, C.H.J. van 2023
Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest solid tumors and is resistant to immunotherapy. B cells play an essential role in PDAC progression and immune responses, both... Show morePancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest solid tumors and is resistant to immunotherapy. B cells play an essential role in PDAC progression and immune responses, both locally and systemically. Moreover, increasing evidence suggests that microbial compositions inside the tumor, as well as in the oral cavity and the gut, are important factors in shaping the PDAC immune landscape. However, the gut-associated lymphoid tissue (GALT) has not previously been explored in PDAC patients. In this study, we analyzed healthy vermiform appendix (VA) from 20 patients with PDAC and 32 patients with colon diseases by gene expression immune profiling, flow cytometry analysis, and microbiome sequencing. We show that the VA GALT of PDAC patients exhibits markers of increased inflammation and cytotoxic cell activity. In contrast, B cell function is decreased in PDAC VA GALT based on gene expression profiling; B cells express significantly fewer MHC class II surface receptors, whereas plasma cells express the immune checkpoint molecule HLA-G. Additionally, the vermiform appendix microbiome of PDAC patients is enriched with Klebsiella pneumoniae, Bifidobacterium animalis, and Adlercreutzia equolifaciens, while certain commensals are depleted. Our findings may suggest impaired B cell function within the GALT of PDAC patients, which could potentially be linked to microbial dysbiosis. Additional investigations are imperative to validate our observations and explore these potential targets of future therapies. Show less
The immune checkpoint VISTA is upregulated on endothelial cells in cancer and selectively prevents T-cell extravasation into tumors. The data highlight a mechanism behind T-cell exclusion and... Show moreThe immune checkpoint VISTA is upregulated on endothelial cells in cancer and selectively prevents T-cell extravasation into tumors. The data highlight a mechanism behind T-cell exclusion and provide a deeper understanding of factors shaping the tumor immune microenvironment.Cancers evade T-cell immunity by several mechanisms such as secretion of anti-inflammatory cytokines, down regulation of antigen presentation machinery, upregulation of immune checkpoint molecules, and exclusion of T cells from tumor tissues. The distribution and function of immune checkpoint molecules on tumor cells and tumor-infiltrating leukocytes is well established, but less is known about their impact on intratumoral endothelial cells. Here, we demonstrated that V-domain Ig suppressor of T-cell activation (VISTA), a PD-L1 homolog, was highly expressed on endothelial cells in synovial sarcoma, subsets of different carcinomas, and immune-privileged tissues. We created an ex vivo model of the human vasculature and demonstrated that expression of VISTA on endothelial cells selectively prevented T-cell transmigration over endothelial layers under physiologic flow conditions, whereas it does not affect migration of other immune cell types. Furthermore, endothelial VISTA correlated with reduced infiltration of T cells and poor prognosis in metastatic synovial sarcoma. In endothelial cells, we detected VISTA on the plasma membrane and in recycling endosomes, and its expression was upregulated by cancer cell-secreted factors in a VEGF-A-dependent manner. Our study reveals that endothelial VISTA is upregulated by cancer-secreted factors and that it regulates T-cell accessibility to cancer and healthy tissues. This newly identified mechanism should be considered when using immunotherapeutic approaches aimed at unleashing T cell-mediated cancer immunity. Show less
Background This study investigates sex disparities in clinical outcomes and tumour immune profiles in patients with pancreatic ductal adenocarcinoma (PDAC) who underwent upfront resection or... Show moreBackground This study investigates sex disparities in clinical outcomes and tumour immune profiles in patients with pancreatic ductal adenocarcinoma (PDAC) who underwent upfront resection or resection preceded by gemcitabine-based neoadjuvant chemoradiotherapy (nCRT).Methods Patients originated from the PREOPANC randomised controlled trial. Upfront surgery was performed in 82 patients, and 66 received nCRT before resection. The impact of sex on overall survival (OS) was investigated using Cox proportional hazards models. The immunological landscape within the tumour microenvironment (TME) was mapped using transcriptomic and spatial proteomic profiling.Results The 5-year OS rate differed between the sexes following resection preceded by nCRT, with 43% for women compared with 22% for men. In multivariate analysis, the female sex was a favourable independent prognostic factor for OS only in the nCRT group (HR 0.19; 95% CI 0.07 to 0.52). Multivariate heterogeneous treatment effects analysis revealed a significant interaction between sex and treatment, implying increased nCRT efficacy among women with resected PDAC. The TME of women contained fewer protumoural CD163+MRC1+M2 macrophages than that of men after nCRT, as indicated by transcriptomic and validated using spatial proteomic profiling.Conclusion PDAC tumours of women are more sensitive to gemcitabine-based nCRT, resulting in longer OS after resection compared with men. This may be due to enhanced immunity impeding the infiltration of protumoral M2 macrophages into the TME. Our findings highlight the importance of considering sex disparities and mitigating immunosuppressive macrophage polarisation for personalised PDAC treatment. Show less
BACKGROUND & AIMS: Improving clinical management of early stage colorectal cancers (T1CRCs) requires a better understanding of their underlying biology. Accumulating evidence shows that cancer... Show moreBACKGROUND & AIMS: Improving clinical management of early stage colorectal cancers (T1CRCs) requires a better understanding of their underlying biology. Accumulating evidence shows that cancer-associated fibroblasts (CAFs) are important determinants of tumor progression in advanced colorectal cancer (CRC), but their role in the initial stages of CRC tumorigenesis is unknown. Therefore, we investigated the contribution of T1CAFs to early CRC progression. METHODS: Primary T1CAFs and patient-matched normal fibroblasts (NFs) were isolated from endoscopic biopsy specimens of histologically confirmed T1CRCs and normal mucosa, respectively. The impact of T1CAFs and NFs on tumor behavior was studied using 3-dimensional co-culture systems with primary T1CRC organoids and extracellular matrix (ECM) remodeling assays. Whole-transcriptome sequencing and gene silencing were used to pinpoint mediators of T1CAF functions. RESULTS: In 3-dimensional multicellular cultures, matrix invasion of T1CRC organoids was induced by T1CAFs, but not by matched NFs. Enhanced T1CRC invasion was accompanied by T1CAF-induced ECM remodeling and up-regulation of CD44 in epithelial cells. RNA sequencing of 10 NF-T1CAF pairs revealed 404 differentially expressed genes, with significant enrichment for ECM-related pathways in T1CAFs. Cathepsin H, a cysteine-type protease that was specifically up-regulated in T1CAFs but not in fibroblasts from premalignant lesions or advanced CRCs, was identified as a key factor driving matrix remodeling by T1CAFs. Finally, we showed high abundance of cathepsin H-expressing T1CAFs at the invasive front of primary T1CRC sections. CONCLUSIONS: Already in the earliest stage of CRC, cancer cell invasion is promoted by CAFs via direct interactions with epithelial cancer cells and stage-specific, cathepsin H-dependent ECM remodeling. RNA sequencing data of the 10 NF-T1CAF pairs can be found under GEO accession number GSE200660. Show less
The lack of multi-omics cancer datasets with extensive follow-up information hinders the identification of accurate biomarkers of clinical outcome. In this cohort study, we performed comprehensive... Show moreThe lack of multi-omics cancer datasets with extensive follow-up information hinders the identification of accurate biomarkers of clinical outcome. In this cohort study, we performed comprehensive genomic analyses on fresh-frozen samples from 348 patients affected by primary colon cancer, encompassing RNA, whole-exome, deep T cell receptor and 16S bacterial rRNA gene sequencing on tumor and matched healthy colon tissue, complemented with tumor whole-genome sequencing for further microbiome characterization. A type 1 helper T cell, cytotoxic, gene expression signature, called Immunologic Constant of Rejection, captured the presence of clonally expanded, tumor-enriched T cell clones and outperformed conventional prognostic molecular biomarkers, such as the consensus molecular subtype and the microsatellite instability classifications. Quantification of genetic immunoediting, defined as a lower number of neoantigens than expected, further refined its prognostic value. We identified a microbiome signature, driven by Ruminococcusbromii, associated with a favorable outcome. By combining microbiome signature and Immunologic Constant of Rejection, we developed and validated a composite score (mICRoScore), which identifies a group of patients with excellent survival probability. The publicly available multi-omics dataset provides a resource for better understanding colon cancer biology that could facilitate the discovery of personalized therapeutic approaches. Show less
BACKGROUND AND AIMS\nMETHODS\nRESULTS\nCONCLUSION\nImproving clinical management of early-stage colorectal cancers (T1CRCs) requires a better understanding of their underlying biology. Accumulating... Show moreBACKGROUND AND AIMS\nMETHODS\nRESULTS\nCONCLUSION\nImproving clinical management of early-stage colorectal cancers (T1CRCs) requires a better understanding of their underlying biology. Accumulating evidence shows that cancer-associated fibroblasts (CAFs) are important determinants of tumor progression in advanced colorectal cancer (CRC), but their role in the initial stages of CRC tumorigenesis is unknown. Therefore, we investigated the contribution of T1CAFs to early CRC progression.\nPrimary T1CAFs and patient-matched normal fibroblasts (NFs) were isolated from endoscopic biopsies of histologically confirmed T1CRCs and normal mucosa, respectively. The impact of T1CAFs and NFs on tumor behavior was studied using 3D co-culture systems with primary T1CRC organoids and extracellular matrix (ECM) remodeling assays. Whole transcriptome sequencing and gene silencing were used to pinpoint mediators of T1CAF functions.\nIn 3D multicellular cultures, matrix invasion of T1CRC organoids was induced by T1CAFs, but not by matched NFs. Enhanced T1CRC invasion was accompanied by T1CAF-induced ECM remodeling and upregulation of CD44 in epithelial cells. RNA sequencing of 10 NF-T1CAF pairs revealed 404 differentially expressed genes, with significant enrichment for ECM-related pathways in T1CAFs. Cathepsin H, a cysteine-type protease that was specifically upregulated in T1CAFs but not in fibroblasts from pre-malignant lesions or advanced CRCs, was identified as a key factor driving matrix remodeling by T1CAFs. Finally, we showed high abundance of Cathepsin H-expressing T1CAFs at the invasive front of primary T1CRC sections.\nAlready in the earliest stage of CRC, cancer cell invasion is promoted by CAFs via direct interactions with epithelial cancer cells and stage-specific, Cathepsin H-dependent ECM remodeling. Show less
Guo, N.N.; Li, N.; Jia, L.; Jiang, Q.Y.; Schreurs, M.; Unen, V. van; ... ; Koning, F. 2023
T he intestine represents the largest immune compartment in the human body, yet its development and organisation during human foetal development is largely unknown. Here we show the immune subset... Show moreT he intestine represents the largest immune compartment in the human body, yet its development and organisation during human foetal development is largely unknown. Here we show the immune subset composition of this organ during development, by longitudinal spectral flow cytometry analysis of human foetal intestinal samples between 14 and 22 weeks of gestation. At 14 weeks, the foetal intestine is mainly populated by myeloid cells and three distinct CD3–CD7+ ILC, followed by rapid appearance of adaptive CD4+, CD8+ T and B cell subsets. Imaging mass cytometry identifies lymphoid follicles from week 16 onwards in a villus-like structure covered by epithelium and confirms the presence of Ki-67+ cells in situ within all CD3–CD7+ ILC, T, B and myeloid cell subsets. Foetal intestinal lymphoid subsets are capable of spontaneous proliferation in vitro. IL-7 mRNA is detected within both the lamina propria and the epithelium and IL-7 enhances proliferation of several subsets in vitro. Overall, these observations demonstrate the presence of immune subset-committed cells capable of local proliferation in the developing human foetal intestine, likely contributing to the development and growth of organized immune structures throughout most of the 2nd trimester, which might influence microbial colonization upon birth. Show less
Bulk, J. van den; Ploeg, M. van der; Ijsselsteijn, M.E.; Ruano, D.; Breggen, R. van der; Duhen, R.; ... ; Miranda, N.F.C.C. de 2023
Background Expression of CD103 and CD39 has been found to pinpoint tumor-reactive CD8+ T cells in a variety of solid cancers. We aimed to investigate whether these markers specifically identify... Show moreBackground Expression of CD103 and CD39 has been found to pinpoint tumor-reactive CD8+ T cells in a variety of solid cancers. We aimed to investigate whether these markers specifically identify neoantigen-specific T cells in colorectal cancers (CRCs) with low mutation burden.Experimental design Whole-exome and RNA sequencing of 11 mismatch repair-proficient (MMR-proficient) CRCs and corresponding healthy tissues were performed to determine the presence of putative neoantigens. In parallel, tumor-infiltrating lymphocytes (TILs) were cultured from the tumor fragments and, in parallel, CD8+ T cells were flow-sorted from their respective tumor digests based on single or combined expression of CD103 and CD39. Each subset was expanded and subsequently interrogated for neoantigen-directed reactivity with synthetic peptides. Neoantigen-directed reactivity was determined by flow cytometric analyses of T cell activation markers and ELISA-based detection of IFN-γ and granzyme B release. Additionally, imaging mass cytometry was applied to investigate the localization of CD103+CD39+ cytotoxic T cells in tumors.Results Neoantigen-directed reactivity was only encountered in bulk TIL populations and CD103+CD39+ (double positive, DP) CD8+ T cell subsets but never in double-negative or single-positive subsets. Neoantigen-reactivity detected in bulk TIL but not in DP CD8+ T cells could be attributed to CD4+ T cells. CD8+ T cells that were located in direct contact with cancer cells in tumor tissues were enriched for CD103 and CD39 expression.Conclusion Coexpression of CD103 and CD39 is characteristic of neoantigen-specific CD8+ T cells in MMR-proficient CRCs with low mutation burden. The exploitation of these subsets in the context of adoptive T cell transfer or engineered T cell receptor therapies is a promising avenue to extend the benefits of immunotherapy to an increasing number of CRC patients. Show less
Vries, N.L. de; Haar, J. van de; Veninga, V.; Chalabi, M.; Ijsselsteijn, M.E.; Ploeg, M. van der; ... ; Voest, E.E. 2023
DNA mismatch repair-deficient (MMR-d) cancers present an abundance of neoantigens that is thought to explain their exceptional responsiveness to immune checkpoint blockade (ICB)(1,2). Here, in... Show moreDNA mismatch repair-deficient (MMR-d) cancers present an abundance of neoantigens that is thought to explain their exceptional responsiveness to immune checkpoint blockade (ICB)(1,2). Here, in contrast to other cancer types(3-5), we observed that 20 out of 21 (95%) MMR-d cancers with genomic inactivation of beta 2-microglobulin (encoded by B2M) retained responsiveness to ICB, suggesting the involvement of immune effector cells other than CD8(+) T cells in this context. We next identified a strong association between B2M inactivation and increased infiltration by gamma delta T cells in MMR-d cancers. These gamma delta T cells mainly comprised the V delta 1 and V delta 3 subsets, and expressed high levels of PD-1, other activation markers, including cytotoxic molecules, and a broad repertoire of killer-cell immunoglobulin-like receptors. In vitro, PD-1(+) gamma delta T cells that were isolated from MMR-d colon cancers exhibited enhanced reactivity to human leukocyte antigen (HLA)-class-I-negative MMR-d colon cancer cell lines and B2M-knockout patient-derived tumour organoids compared with antigen-presentation-proficient cells. By comparing paired tumour samples from patients with MMR-d colon cancer that were obtained before and after dual PD-1 and CTLA-4 blockade, we found that immune checkpoint blockade substantially increased the frequency of gamma delta T cells in B2M-deficient cancers. Taken together, these data indicate that gamma delta T cells contribute to the response to immune checkpoint blockade in patients with HLA-class-I-negative MMR-d colon cancers, and underline the potential of gamma delta T cells in cancer immunotherapy. Show less
Vries, N.L. de; Haar, J. van de; Veninga, V.; Chalabi, M.; Ijsselsteijn, M.E.; Ploeg, M. van der; ... ; Voest, E.E. 2023
DNA mismatch repair-deficient (MMR-d) cancers present an abundance of neoantigens that is thought to explain their exceptional responsiveness to immune checkpoint blockade (ICB)1,2. Here, in... Show moreDNA mismatch repair-deficient (MMR-d) cancers present an abundance of neoantigens that is thought to explain their exceptional responsiveness to immune checkpoint blockade (ICB)1,2. Here, in contrast to other cancer types3,4,5, we observed that 20 out of 21 (95%) MMR-d cancers with genomic inactivation of β2-microglobulin (encoded by B2M) retained responsiveness to ICB, suggesting the involvement of immune effector cells other than CD8+ T cells in this context. We next identified a strong association between B2M inactivation and increased infiltration by γδ T cells in MMR-d cancers. These γδ T cells mainly comprised the Vδ1 and Vδ3 subsets, and expressed high levels of PD-1, other activation markers, including cytotoxic molecules, and a broad repertoire of killer-cell immunoglobulin-like receptors. In vitro, PD-1+ γδ T cells that were isolated from MMR-d colon cancers exhibited enhanced reactivity to human leukocyte antigen (HLA)-class-I-negative MMR-d colon cancer cell lines and B2M-knockout patient-derived tumour organoids compared with antigen-presentation-proficient cells. By comparing paired tumour samples from patients with MMR-d colon cancer that were obtained before and after dual PD-1 and CTLA-4 blockade, we found that immune checkpoint blockade substantially increased the frequency of γδ T cells in B2M-deficient cancers. Taken together, these data indicate that γδ T cells contribute to the response to immune checkpoint blockade in patients with HLA-class-I-negative MMR-d colon cancers, and underline the potential of γδ T cells in cancer immunotherapy. Show less
Roelands, J.; Ploeg, M. van der; Ijsselsteijn, M.E.; Dang, H.; Boonstra, J.J.; Hardwick, J.C.H.; ... ; Miranda, N.F.C.C. de 2022
ObjectiveBiological insights into the stepwise development and progression of colorectal cancer (CRC) are imperative to develop tailored approaches for early detection and optimal clinical... Show moreObjectiveBiological insights into the stepwise development and progression of colorectal cancer (CRC) are imperative to develop tailored approaches for early detection and optimal clinical management of this disease. Here, we aimed to dissect the transcriptional and immunologic alterations that accompany malignant transformation in CRC and to identify clinically relevant biomarkers through spatial profiling of pT1 CRC samples. DesignWe employed digital spatial profiling (GeoMx) on eight pT1 CRCs to study gene expression in the epithelial and stromal segments across regions of distinct histology, including normal mucosa, low-grade and high-grade dysplasia and cancer. Consecutive histology sections were profiled by imaging mass cytometry to reveal immune contextures. Finally, publicly available single-cell RNA-sequencing data was analysed to determine the cellular origin of relevant transcripts. ResultsComparison of gene expression between regions within pT1 CRC samples identified differentially expressed genes in the epithelium (n=1394 genes) and the stromal segments (n=1145 genes) across distinct histologies. Pathway analysis identified an early onset of inflammatory responses during malignant transformation, typified by upregulation of gene signatures such as innate immune sensing. We detected increased infiltration of myeloid cells and a shift in macrophage populations from pro-inflammatory HLA-DR(+)CD204(-) macrophages to HLA-DR(-)CD204(+) immune-suppressive subsets from normal tissue through dysplasia to cancer, accompanied by the upregulation of the CD47/SIRP alpha 'don't eat me signal'. ConclusionSpatial profiling revealed the molecular and immunological landscape of CRC tumourigenesis at early disease stage. We identified biomarkers with strong association with disease progression as well as targetable immune processes that are exploitable in a clinical setting. Show less
Background: The complete response rate of cervical high-grade squamous intraepithelial lesion (cHSIL) patients to imiquimod immunotherapy is approximately 60%. Consequently, many patients are... Show moreBackground: The complete response rate of cervical high-grade squamous intraepithelial lesion (cHSIL) patients to imiquimod immunotherapy is approximately 60%. Consequently, many patients are exposed to unnecessary adverse effects of imiquimod. On the other hand, conventional surgical large loop excision therapy is associated with increased risk of premature births in subsequent pregnancies. An in-depth analysis of the cHSIL immune microenvironment was performed in order to identify and develop a predictive biomarker for response to imiquimod, to maximize therapy efficacy and to avoid adverse effects in patients unlikely to respond. Methods: Biopsies of 35 cHSIL patients, before and 10 weeks on imiquimod treatment, were analyzed by two multispectral seven-color immunofluorescence panels for T cell and myeloid cell composition in relation to treatment response. Based on these results a simplified immunohistochemical detection protocol was developed. Samples were scanned with the Vectra multispectral imaging system and cells were automatically identified using machine learning. Results: The immune microenvironment of complete responders (CR) is characterized by a strong and coordinated infiltration by T helper cells (activated PD1(+)/type 1 Tbet(+)), M1-like macrophages (CD68(+)CD163(-)) and dendritic cells (CD11c(+)) prior to imiquimod. The lesions of non-responders (NRs) displayed a high infiltration by CD3(+)FOXP3(+) regulatory T cells. At 10 weeks on imiquimod, a strong influx of intraepithelial and stromal CD4(+) T cells was observed in CR but not NR patients. A steep decrease in macrophages occurred both in CR and NR patients, leveling the pre-existing differences in myeloid cell composition between the two groups. Based on the pre-existing immune composition differences, the sum of intraepithelial CD4 T cell, macrophage and dendritic cell counts was used to develop a quantitative simplified one color immunohistochemical biomarker, the CHSIL immune biomarker for imiquimod (CIBI), which can be automatically and unbiasedly quantified and has an excellent predictive capacity (receiver operating characteristic area under the curve 0.95, p<0.0001). Conclusion: The capacity of cHSIL patients to respond to imiquimod is associated with a pre-existing coordinated local immune process, fostering an imiquimod-mediated increase in local T cell infiltration. The CIBI immunohistochemical biomarker has strong potential to select cHSIL patients with a high likelihood to experience a complete response to imiquimod immunotherapy. Show less
Hos, B.J.; Tondini, E.; Camps, M.G.M.; Rademaker, W.; Bulk, J. van den; Ruano, D.; ... ; Ossendorp, F. 2022
We report an approach to identify tumor-specific CD4+ T cell neo-epitopes of both mouse and human cancer cells by analysis of major histocompatibility complex (MHC) class II-eluted natural peptides... Show moreWe report an approach to identify tumor-specific CD4+ T cell neo-epitopes of both mouse and human cancer cells by analysis of major histocompatibility complex (MHC) class II-eluted natural peptides. MHC class II -presented peptide sequences are identified by introducing the MHC class II transactivator (CIITA) in tumor cells that were originally MHC class II negative. CIITA expression facilitates cell-surface expression of MHC class II molecules and the appropriate peptide-loading machinery. Peptide elution of purified MHC class II molecules and subsequent mass spectrometry reveals oncoviral-and neo-epitopes as well as shared epitopes. Immunological relevance of these epitopes is shown by natural presentation by dendritic cells and immunogenicity. Synthetic peptide vaccination induced functional CD4+ T cell responses, which helped tu-mor control in vivo. Thus, this CIITA transfection approach aids to identify relevant T helper epitopes pre-sented by any MHC class II allele that would be otherwise very difficult to predict and reveals important tar-gets for cancer immunotherapy. Show less
Hos, B.J.; Tondini, E.; Camps, M.G.M.; Rademaker, W.; Bulk, J. van den; Ruano, D.; ... ; Ossendorp, F. 2022
We report an approach to identify tumor-specific CD4+ T cell neo-epitopes of both mouse and human cancer cells by analysis of MHC class II-eluted natural peptides. MHC class II-presented peptide... Show moreWe report an approach to identify tumor-specific CD4+ T cell neo-epitopes of both mouse and human cancer cells by analysis of MHC class II-eluted natural peptides. MHC class II-presented peptide sequences are identified by introducing the MHC class II transactivator CIITA in tumor cells that were originally MHC class II-negative. CIITA expression facilitates cell-surface expression of MHC class II molecules and the appropriate peptide-loading machinery. Peptide elution of purified MHC class II molecules and subsequent mass spectrometry reveals many novel oncoviral-, shared and neo-epitopes. Immunological relevance of these epitopes is shown by natural presentation by dendritic cells and immunogenicity. Synthetic peptide vaccination induced functional CD4+ T cell responses which helped tumor control in vivo. Thus, this CIITA transfection approach aids to identify relevant T helper epitopes presented by any MHC class II allele that would be otherwise very difficult to predict and reveals important new targets for cancer immunotherapy. Show less
Brouwer, T.P.; Zanden, S.Y. van der; Ploeg, M. van der; Eendenburg, J.D.H. van; Bonsing, B.A.; Miranda, N.F.C.C. de; ... ; Vahrmeijer, A.L. 2022
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of cancer, mainly due to its delayed diagnosis and lack of effective therapeutic options. Therefore, it is imperative to find... Show morePancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of cancer, mainly due to its delayed diagnosis and lack of effective therapeutic options. Therefore, it is imperative to find novel treatment options for PDAC. Here, we tested a series of conventional chemotherapeutics together with anthracycline compounds as single agents or in combination, determining their effectivity against established commercial and patient-derived, low-passage PDAC cell lines. Proliferation and colony formation assays were performed to determine the anticancer activity of anthracyclines; aclarubicin and doxorubicin, on commercial and patient-derived, low-passage PDAC cell lines. In addition, the effect of standard-of-care drugs gemcitabine and individual components of FOLFIRINOX were also investigated. To evaluate which mechanisms of cell death were involved in drug response, cleavage of poly(ADP-ribose)polymerase was evaluated by western blot. Aclarubicin showed superior antitumor activity compared to other anthracyclines and standard of care drugs (gemcitabine and individual components of FOLFIRINOX) in a patient-derived, low-passage PDAC cell line and in commercial cell lines. Importantly, the combination of gemcitabine and aclarubicin showed a synergistic effect at a dose range where the single agents by themselves were ineffective. In parallel, evaluation of the antitumor activity of aclarubicin demonstrated an apoptotic effect in all PDAC cell lines. Aclarubicin is cytotoxic for commercial and patient-derived low-passage PDAC cell lines, at doses lower than peak serum concentrations for patient treatment. Our findings support a (re)consideration of aclarubicin as a backbone of new combination regimens for pancreatic cancer patients. Show less
