Jitka Fucikova

Relevant pre-clinical models are essential for driving progress in cancer therapy research. Here, we develop a pre-clinical study framework using an injectable orthotopic lung adenocarcinoma (LUAD) model (ORTHO) that replicates key features of human LUAD patients and is dissectible into tumoural and non-tumoural adjacent tissue, in analogy with patient samples. We also present SEPARATE-Seq, a broadly applicable technique enabling the partitioning of vascular and intratissue immune cells along with scRNA-Seq. By applying both SEPARATE-Seq and spatial transcriptomics to our dissectible ORTHO model, we confirm that our model replicates key immune features of human LUAD patients. Similarly to these patients, we observe NK-cell dysfunction and neutrophil dichotomy, and show that these are affected by their vascular/intratissue or tumour/adjacent location, highlighting the need for these spatial distinctions. Additionally, we show that several immune populations are restricted to specialised, local niches within the tumour, including a ring of lipid-associated TAMs lining the tumour edge and hubs of interferon-stimulated cells. Overall, our resource, available through an interactive tool, provides a comprehensive multiomics immune characterisation of a reproducible pre-clinical LUAD mouse model.

Natural killer (NK) cells contribute to tumor immunosurveillance, yet their heterogeneity across cancer types remains incompletely understood. Transcriptomic, spatial, and functional assays reveal that non-small cell lung carcinoma (NSCLC) is enriched in NK cells that mediate clinically relevant effector functions, whereas high-grade serous ovarian carcinoma (HGSOC) contains dysfunctional NK cells that express co-inhibitory receptors including NKG2A. Analysis of HGSOC patient samples and syngeneic mouse models indicates a crosstalk between NK cells and CD8⁺ T cells critical for effective antitumor immunity. Depletion of either population leads to phenotypic impairment of the reciprocal one. Blocking NKG2A restores NK cell cytotoxicity and promotes CD8⁺ T cell responses, significantly improving the efficacy of PD-1 blockade in murine HGSOC models. Thus, NK cells and CD8⁺ T cells engage in a functional interplay of immunological relevance. Moreover, the NKG2A–HLA-E axis represents a clinically actionable immunological checkpoint in tumors with impaired NK cell functions.

The omentum, a visceral adipose tissue with critical metabolic, immunological, and stem cell functions is the preferred site for ovarian cancer metastasis. However, its role in maintaining homeostasis and its responses to metastatic colonization remain incompletely understood. Using single-cell transcriptomics, we profile different anatomical regions of the omentum in patients with benign conditions and metastasis. We catalog the benign omentum and observe a stable cell type composition and a preserved stem and progenitor niche. Upon metastatic colonization, we report on increased immune heterogeneity and a concomitant reduction of mesothelial and progenitor cells. The lesser omentum, which is not routinely removed during surgical debulking, is identified as a premetastatic niche characterized by neutrophil infiltration, extracellular trap formation, and the presence of micrometastases. At established metastatic sites, resident cells exhibit cancer-associated phenotypes with regulatory, anti-adipogenic, and immunosuppressive properties. Cellular reprogramming across the omentum is associated with signaling profiles of tumor cells, suggesting potential influences on both proximal and distal tissue regions. This cell atlas illuminates the cellular and molecular determinants of organ homeostasis and reveals a high degree of plasticity and cellular reprogramming promoted by cancer colonization.

The clinical relevance of combined chemotherapy and immunotherapy in mouse models of TMBLo and TMBHi ovarian cancer. A and B, Bar plots showing single-nucleotide variants positions (SNVs) (A) and the somatic mutations prevalence (mutations per megabase) (B) in ID8 (n = 3) and Brca1−/−Trp53−/−/Myc/Hras SO1 (n = 3) C57BL/6 syngeneic mouse ovarian cancer cell lines. Mean and SEM are shown. Statistical significance was calculated by multiple t test. P values are indicated. C, Experimental design for the analysis of TLS aggregates development and efficacy of combined chemotherapy and aPD1 and/or aTIM-3 therapy in TMBLo ID8 and TMBHi SO1 experimental syngeneic mouse models. D and E, Representative immunostaining for CD4, CD8, CD20, and CD21 (D) and a box plot showing density of TLS aggregates within chemo-naïve (n = 5) and treated TMBHi SO1 (n = 9) ovarian tumors (E). Scale bars, 100 µm and 2.5 mm. Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated. F–H, Representative dot plot (F) and flow cytometry analyses for percentages of CD62L+CD44+ central memory (CM) and CD62L−CD44− terminally differentiated CD8+ T cells (TEMRA) (G) and TCF1+PD1+CD8+ and TIM-3+PD1+CD8+ T cells (H) in tumor samples of the TMBHi SO1 experimental model in the presence or absence of carboplatin and taxane chemotherapy (NACT). Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated. I and J, Overall survival (OS) of TMBHi SO1 experimental model (I) and flow cytometry analyses for percentage of TCF1+PD1+CD8+ T cells after NACT, aPD1, aTIM-3 and combined therapy (J). Survival curves were estimated by the Kaplan–Meier method, and differences between groups were evaluated using log-rank test. Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated. K and L, Representative immunostaining for CD4, CD8, CD20, and CD21 (K) and a box plot showing density of TLS aggregates within chemo-naïve (n = 8) and treated TMBLo ID8 (n = 8) (L) ovarian tumors. Scale bars, 100 µm and 2.5 mm. Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated. M, Flow cytometry analyses for TCF1+PD1+CD8+ and TIM-3+PD1+CD8+ T cells in tumor samples of the TMBLo ID8 experimental model in the presence or absence of carboplatin and taxane chemotherapy (NACT). Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated. N, Overall survival (OS) of TMBLo ID8 experimental model after NACT, anti-PD1, and combined therapy. Survival curves were estimated by the Kaplan–Meier method, and differences between groups were evaluated using log-rank test. P values are indicated. (Panel C created with BioRender.com.)

NACT-mediated progenitor TCF1+PD1+CD8+ T-cell phenotype associates with effector cytotoxic functions within metastatic HGSOC. A–C, Representative dot plot (A) and box plot (B) showing percentage of TIM-3+PD1+CD8+ T cells and GZMB+CD8+ T cells within native pTME and mTME of chemo-naïve and treated HGSOC as determined by flow cytometry. Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. Statistical significance was calculated by the Mann–Whitney test. P values are indicated. C, Marker heatmap dot plots obtained after t-SNE and showing the relative expression of the indicated marker in the different phenotypic clusters within mTME of chemo-naïve and treated HGSOS as determined by flow cytometry. D, Design of experimental and sequencing workflow in 11 patients with HGSOC before and after NACT. scRNAseq was performed on dissociated solid tumor specimens using 10× Genomics Chromium platform. E and F, Uniform manifold approximation and projection (UMAP) plot of all cells (n = 51,476) passing the quality control, colored by type of therapy (E) and cell type (F). G and H, TILs projections (G) and predicted subtype frequencies (H) in biopsies from patients with chemo-naïve and treated HGSOC. CM, central memory; EM, effector memory; MAIT, mucosal-associated invariant T cells; PTEX, progenitor exhausted T cells; TEMRA, terminally exhausted T cells; TEX, exhausted T cells. I and J, Radar plot showing percentage of CD8+ T cells expressing respective T-cell marker (KLRB1, TCF7, CCR7, IL7R, LMNA, FGFBP2, XCL1, CD200, CRTAM, TOX, PDCD1, HAVCR2, and GNLY; I) and UMAP showing expression of PDCD1, HAVCR2 and TCF7 in CD4+ and CD8+ T-cell clusters in chemo-naïve and treated HGSOC samples (study cohort 5; J), as determined by scRNA-seq. (Panel D created with BioRender.com.)

NACT-mediated adjuvanticity positively impacts clinically relevant TLS maturation in metastatic HGSOC. A, Representative image of immunofluorescence of CD4, CD8, CD20, CD21, CD23, DC-LAMP, and GZMB staining (immunofluorescence panel 1). Scale bars, 10, 100 and 500 µm. B and C, Distribution of early TLS (eTLS; B) and mature TLS (mTLS; C) across pTME and mTME HGSOC tumor samples with/without NACT. Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated. D, Supervised hierarchical clustering of TLS-relevant gene signature (CCL2, CCL3, CCL4, CCL5, CCL8, CCL18, CCL19, CCL21, CXCL9, CXCL10, CXCL11, and CXCL13) across pTME and mTME HGSOC tumor samples with/without NACT. E and F, Overall survival (OS) of 60 (E) and 40 chemo-naïve and treated patients with mHGSOC (F), respectively (study cohort 1 and 2) based on median stratification of total mTLS. Survival curves were estimated by the Kaplan–Meier method, and differences between groups were evaluated using log-rank test. Number of patients at risk and P values are reported. G, Number of mTLS across patients with CALRLo and CALRHi mHGSOC with/without NACT as determined by median stratification. Mean and SEM are shown. Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated. H and I, OS of 60 and 40 patients with chemo-naïve and treated mHGSOC (study cohorts 1 and 2), upon stratification based on median frequency of mTLS and expression of CALR. Survival curves were estimated by the Kaplan–Meier method, and differences between groups were evaluated using the log-rank test.

Immunomodulation by NACT in metastatic HGSOC. A, Representative images of CALR immunostaining in CALRLo and CALRHi patients. Scale bars, 10 and 100 µm. B, CALR expression levels determined by immunostaining and (C) ER stress signature level [expression level of DNA damage inducible transcript 3 (DDIT3, best known as CHOP), heat shock protein family A (Hsp70) member 5 (HSPA5, best known as BIP), and heat shock protein 90 beta family member 1 (HSP90B1)] as determined by RNA-seq in pTME and mTME HGSOC with/without NACT. Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated. D, Supervised hierarchical clustering of gene signatures related to immune populations (orange), immune functions (blue) and immune phenotype (purple) as determined by RNA-seq data from pTME and mTME HGSOC tumor samples with/without NACT. IS, immunosuppression; mDCs, myeloid dendritic cells; NK cells, natural killer cells; TLS, tertiary lymphoid structures. E, Gene expression signature associated with CD8+ T cells, B cells, cytotoxicity, mDCs, TLS, and immunosuppression as determined on RNA-seq data from pTME and mTME HGSOC with/without NACT. Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated. F, Representative image of CD20/DC-LAMP double immunostaining. Scale bars, 500 and 100 µm. G, Density of CD8+ T cells, CD20+ B cells, and DC-LAMP+ cells as determined by immunostaining in pTME and mTME HGSOC samples with/without NACT. Mean and SEM are shown. Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated. ns, not significant.

NACT positively increases the ICI-sensitive TCF1+PD1+CD8+ T-cell phenotype within metastatic HGSOC. A, Representative image of immunofluorescence of CD68, CD8, PD-L1, FoxP3, TCF1, CD57, PanCK, PD1, CD4, CD20, GZMB, and TIM-3 staining (immunofluorescence panel 2). Scale bars, 2 μm, 10 µm, and 100 µm. B, Violin plot showing the density of CD8+ and PD1+CD8+ within tumor core and tumor stroma of pTME and mTME of chemo-naïve and treated HGSOC. Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated. C and D, Representative image (C) and box plot showing the density of TCF1+PD1+CD8+ T cells and TIM-3+PD1+CD8+ and spatial distribution of TCF1+PD1+CD8+ T cells within tumor core and stroma in pTME and mTME of chemo-naïve and treated HGSOC (D). Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated. E, Supervised hierarchical clustering of gene signatures associated with different stages of T-cell differentiation: T-cell stemness (orange), T-cell effector function (blue), T-cell proliferation (green), T-cell phenotype (red) as determined by RNAseq in mTME of chemo-naïve and treated HGSOC. For further details, see Supplementary Fig. S9. F and G, Representative image of digital pathology spatial distribution and violin plot showing the number of cell contacts between PanCK+ malignant cells and TCF1+PD1+CD8+ T cells and TIM-3+PD1+CD8+ within 0 to 30 µm in pTME and mTME of chemo-naïve and treated HGSOC.

Univariate Cox proportional hazard analyses.

NACT-mediated adjuvanticity positively impacts the density of follicular T cells (TFH) and in situ activation of intratumoral B cells in mHGSOC. A–C, Representative image (A) and box plots showing the density of CD4+ cells (B) and CXCR5+PD1+FoxP3−CD4+ TFH cells (C) in the pTME and mTME of chemo-naïve and treated HGSOC (study cohort 1 and 2). Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. Statistical significance was calculated by the Mann–Whitney test. P values are indicated. D and E, Dot plot (D) and box plot (E) showing expression profile of gene signatures of B-cell subtypes, e.g., plasma cells (PC), germinal center (GC), and memory B cells within pTME and mTME HGSOC tumor samples with/without NACT. Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated. F and G, Representative image (F) and density of CD68+CD163+ TAMs in pTME and mTME of chemo naïve and treated HGSOC (G). Mean and SEM are shown. Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated.