Tumoral and stromal hMENA isoforms impact tertiary lymphoid structure localization in lung cancer and predict immune checkpoint blockade response in patients with cancer

Abstract

Background: Tertiary Lymphoid Structures (TLS) correlate with positive outcomes in patients with NSCLC and the efficacy of immune checkpoint blockade (ICB) in cancer. The actin regulatory protein hMENA undergoes tissue-specific splicing, producing the epithelial hMENA^11a linked to favorable prognosis in early NSCLC, and the mesenchymal hMENAΔv6 found in invasive cancer cells and pro-tumoral cancer-associated fibroblasts (CAFs). This study investigates how hMENA isoforms in tumor cells and CAFs relate to TLS presence, localization and impact on patient outcomes and ICB response.

Methods: Methods involved RNA-SEQ on NSCLC cells with depleted hMENA isoforms. A retrospective observational study assessed tissues from surgically treated N0 patients with NSCLC, using immunohistochemistry for tumoral and stromal hMENA isoforms, fibronectin, and TLS presence. ICB-treated patient tumors were analyzed using Nanostring nCounter and GeoMx spatial transcriptomics. Multiparametric flow cytometry characterized B cells and tissue-resident memory T cells (T_RM). Survival and ICB response were estimated in the cohort and validated using bioinformatics pipelines in different datasets.

Findings: Findings indicate that hMENA^11a in NSCLC cells upregulates the TLS regulator LTβR, decreases fibronectin, and favors CXCL13 production by T_RM. Conversely, hMENAΔv6 in CAFs inhibits LTβR-related NF-kB pathway, reduces CXCL13 secretion, and promotes fibronectin production. These patterns are validated in N0 NSCLC tumors, where hMENA11a^high expression, CAF hMENAΔv6^low, and stromal fibronectin^low are associated with intratumoral TLS, linked to memory B cells and predictive of longer survival. The hMENA isoform pattern, fibronectin, and LTβR expression broadly predict ICB response in tumors where TLS indicates an anti-tumor immune response.

Interpretation: This study uncovers hMENA alternative splicing as an unexplored contributor to TLS-related Tumor Immune Microenvironment (TIME) and a promising biomarker for clinical outcomes and likely ICB responsiveness in N0 patients with NSCLC.

Funding: This work is supported by AIRC (IG 19822), ACC (RCR-2019-23669120), CAL.HUB.RIA Ministero Salute PNRR-POS T4, "Ricerca Corrente" granted by the Italian Ministry of Health.

Keywords: Cancer-associated fibroblasts; Epithelial mesenchymal transition; Fibronectin; Immune checkpoint blockade; Non-small cell lung cancer; Resistance to immunotherapy; Tertiary lymphoid structures; Tumor microenvironment; hMENA isoforms.

Fig. 1

The depletion of hMENA^11adown-regulates LTβR and up-regulates FN1. In node-negative patients with NSCLC, hMENA^11aexpression associates with intratumoral TLS. a. Volcano plots showing differentially expressed genes (q-value <0.05, n = 1096, GSE217451) in Si-hMENA^11a versus control H1650 NSCLC cells (n = 3). Reported are the negative log10-transformed adjusted P values plotted against the log2 fold changes. Dots represent individual genes. b. qRT-PCR analysis of LTβR mRNA expression in the indicated cell lines transfected with control (Si-CNTR), and hMENA^11a pool SiRNAs (Si-hMENA^11a) or with plasmid control (CNTR) and hMENA^11a expressing vector (hMENA^11a). The control of hMENA^11a expression in the transfected cells by WB is reported in Supplementary Figure S3a. Data are reported as the mean ± SD of technical triplicates which are representative of two independent experiments. P value was calculated by 2-tailed Student's t test. c. qRT-PCR analysis showed that LTβR and hMENA^11a mRNA expression level correlate in NSCLC cell lines. The value of Spearman correlation is reported. d. Consecutive sections of a representative NSCLC primary tumor hMENA^11a positive, showing low stromal FN1, TLS IT localization and high CXCL13. Magnification 8×. Scale bar 300 μm. Right, histograms relative to the IHC analysis of 94 node-negative NSCLC tissues showing that hMENA^11a positive cases more frequently show TLS within the tumor area (TLS IT). e. Consecutive sections of a representative case of lung adenocarcinoma hMENA^11a negative, showed high stromal FN1, TLS PT and low CXCL13. Right, histograms are relative to the IHC analysis of 94 node-negative NSCLC tissues showing that hMENA^11a negative (hMENA^{11a low}/hMENA (t)^high) cases more frequently show peritumoral TLS (TLS PT). P value was estimated with Fisher Exact test.

Fig. 2

TLS organized within the tumor core are predictive of survival in 94 early patients with NSCLC. a. Representative case of a lung ADC stained for B (CD20) and T (CD3) cells showing the presence of TLS within the tumor area (IT). b and c. Kaplan–Meier estimate of disease-free survival (DFS) (b) and overall survival (OS) (c) of resected, node-negative patients with NSCLC according to the presence of TLS IT. d. Representative case of an undifferentiated cancer of the lung stained as in A, showing the presence of TLS in the peritumoral region (PT). e and f. Kaplan–Meier estimate of DFS (e) or OS (f) of resected, node-negative patients with NSCLC according to the presence of TLS PT. P value was estimated with log-rank test.

Fig. 3

TLS located within tumor core (IT⁺) are associated with high percentage of switched memory phenotype B cells. a. Representative dot plots showing CD19⁺ (left panel) and CD27 versus IgD staining (right panel), in ex vivo lymphocytes derived from PBMC (P), non-tumoral (N) and tumor tissues (T), of two patients with NSCLC non-possessing (IT-) or possessing (IT+) TLS within the tumor core. Percentages of different subsets are indicated as black numbers. Red numbers represent the proportion of switched (CD27⁺IgD⁻) or unswitched (CD27⁺IgD⁺) subsets within total memory B cells (CD27⁺). b–g. Pooled results from patients with TLS IT⁻ (grey color, n = 4) and TLS IT⁺ (blue color, n = 4) NSCLC showing the percentage of total B cells (CD19⁺) within the lymphocyte gate, and the principal B-cell differentiation subsets within CD19⁺ B cells. Transitional B cells (CD24⁺CD38⁺); Naïve (CD27⁻IgD⁺); Memory B cells (total CD27⁺); Unswitched memory B cells (CD27⁺IgD⁺); Switched memory B cells (CD27⁺IgD⁻). Left panels, comparison between matched P, N and T sites (non-parametric Fridman test), within IT⁻ or IT⁺ group; right panels, comparison between IT⁻ versus IT⁺ within the same site (non-parametric Mann–Whitney test). NS, not significant.

Fig. 4

NSCLC primary tumors with high level of stromal fibronectin display TLS localized in the peritumoral region (PT) and express stromal hMENA(t). a and b. Histograms relative to IHC of 94 node-negative NSCLC tissues showed that high stromal FN1 level associated with TLS PT (a) but not with TLS IT (b). P value was estimated with Fisher Exact test. c. Representative images of multiplex immunofluorescence labeling of two NSCLC cases showing TLS IT (CD20 green) and α-SMA positive stromal cells (red) negative for pan-hMENA staining (yellow) (left) or TLS PT with stromal cells both positive for hMENA staining (right) and α-SMA. Magnification 10×. Zooms of stromal area are indicated by white squares. d. Immunohistochemistry of consecutive sections of representative NSCLC cases with pan-hMENA negative or pan-hMENA positive stromal cells. e–g. Histograms relative to IHC characterization of 54 node-negative NSCLC tissues showed that stromal pan-hMENA positivity is associated with high stromal FN1 level (e), TLS PT positive (f) and TLS IT negative (g). P value was estimated with Fisher Exact test. h. Representative WB analysis of FN1 expression in CAF #377 transfected with control (Si-CNTR), and hMENA(t) pool siRNAs (Si-hMENA(t) (upper panel). Densitometrically quantified data, represented as fold change of FN1/ACTIN ratio with respect to control (Si-CNTR) set as 1 (lower panel). Data are reported as the mean ± SD of 4 different CAF population (#358; #361; #376; #377). P value was calculated by paired 2-tailed Student's t test. i. Representative images of immunofluorescence of fibronectin fibrils (FN-488) in CAFs of patient #361, transfected with non-targeting control (CNTR) and hMENA(t) siRNAs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Magnification 20×. Scale bar: 50 μm.

Fig. 5

hMENA/hMENAΔv6 influences the expression and signaling of LTβR and the secretion of CXCL13 in CAFs. The ‘epithelial’ hMENA^11aisoform in tumor cells affects CXCL13 production by T_RMcells. a. qRT-PCR analysis of LTβR mRNA expression in the CAFs obtained from four different patients with NSCLC (#359, #358, #391, #405), transfected with control (Si-CNTR), and hMENA(t) pool siRNAs (Si-hMENA(t)). Data reported are the mean of technical triplicates. P value of paired 2-tailed Student's t test is reported. b. Representative WB analysis with the indicated Abs of protein extracts from CAF #302 transfected with non-targeting siRNA (Si-CNTR) or with hMENA(t) siRNA, untreated or treated with 50 ng/mL of LIGHT for 24 h. Fold change of P-p65/p65 and of p52/p100 staining intensity (right). Data reported are the mean of 4 different experiments ± SD. Adjusted P values of One-way ANOVA followed by Tukey's multiple comparisons procedures are reported when significant. c. CXCL13 production evaluated by ELISA assay of CAFs derived from three different patients (#302; #358; #571) transfected with non-targeting siRNA (Si-CNTR) or with hMENA(t) siRNA. Data reported are the mean of technical triplicates of pg/mL normalized for total protein content in three biological replicates. P value of paired two tailed t test, is reported d. Percentage of CXCL13 chemokine, evaluated by multiparametric flow cytometry, in ex-vivo TILs isolated from eight patients with NSCLC, after culture (24 h) with CM from H1650 tumor cells silenced for hMENA^11a (Si-hMENA^11a), or control (Si-CNTR). Results within CD8⁺ T_RM (CD103⁺CD69⁺, left panel) or CD4⁺ T_RM (CD103⁺CD69⁺, right panel) are shown. Statistical significance was determined using non-parametric Wilcoxon rank test.

Fig. 6

hMENA11a^high/hMENAΔv6^lowfavors OS in TGCA patients with lung cancer and associate with TLS and low immunosuppressive ECM-myCAF signatures. The pattern of hMENA isoforms impacts ICB response. a. OS curves in early stages patients with LUNG cancer (LUSC and LUAD) from The Cancer Genome Atlas (TCGA). Patients were stratified into two groups, hMENA11a^high/hMENAΔv6^low and hMENAΔv6^high/FN1^high, on the basis of the specific percent spliced-in (PSI) values for exon 6 and 11a and the expression levels for FN1 gene and 11a isoform. Statistical significance was calculated by using the log-rank test. P value is shown. b and c. Barplots showing the percentage of hMENA11a^high/hMENAΔv6^low and hMENAΔv6^high/FN1^high cases defined as in a, stratified according to the classic cytokine-based TLS signature (b) and the Ig-enriched TLS signature (Meylan, c) in combination with the signature of immunosuppressive ECM-myCAF (CAF). The percentage of hMENA11a^high/hMENAΔv6^low and hMENAΔv6^high/FN1^high cases in each group and P values are shown. d. Nanostring analysis of NSCLC tissues from 12 ICB treated patients with high or low levels of hMENA,^11a hMENAΔv6, FN1 and LTβR indicating that tumor tissues of all the poor responder patients (PR) are hMENA11a^low, hMENAΔv6^high, FN1^high and LTβR^low. e. Proportions of patients with melanoma with response to ICB treatment (GR) or with poor response to treatment (PR), stratified by hMENAΔv6 isoform, FN1 and LTβR expression level. f. Proportions of patients with TNBC with response to anti-PD1 treatment (i.e., T cell expanded, ‘E’) or with non-response to treatment (i.e., T cell non-expanded, ‘NE’), stratified by hMENAΔv6 splicing and LTβR expression level. b–f. All P values were estimated using Fisher Exact Test. d–f. N = Number of Patients. g and h. Representative images of NSCLC tissues from PR patient with TLS PT and GR patient with TLS IT, morphologically defined by Pan-CK (green, tumor cells) and CD45 (red, immune cells) obtained by GeoMx DSP platform. i. Volcano plots showing differentially expressed genes (q < 0.05, n = 581) in TLS PT/PR versus TLS IT/GR patients in their tumor cells. Reported are the negative log10-transformed adjusted P values plotted against the log2 fold changes. Dots represent individual genes. Genes of interest are indicated.