Mechanoimmunological Control of Metastatic Site Selection

Abstract

Cancer cells alter their mechanical properties in response to the rigidity of their environment. Here, we explored the implications of this environmental mechanosensing for anti-tumor immunosurveillance using single cell biophysical profiling and metastasis models. Cancer cells stiffened in more rigid environments, a biophysical change that sensitized them to cytotoxic lymphocytes. In immunodeficient mice, this behavior manifested in the outgrowth of stiffer metastatic cells in the rigid bone than in the soft lung, while in immunocompetent hosts, it led to preferential elimination of stiffer cancer cells and suppression of bone metastasis. Environmentally-induced cell stiffening and immune sensitization both required Osteopontin, a secreted glycoprotein that is upregulated during bone colonization. Analysis of patient metastases spanning mechanically distinct tissues revealed associations between environmental rigidity, immune infiltration, and cancer cell stiffness consistent with mechanically driven immunosurveillance. These results demonstrate how environmental mechanosensing modulates anti-tumor immunity and suggest a mechanoimmunological basis for metastatic site selection.

Fig. 1:. Substrate rigidity stiffens cancer cells and sensitizes them to cytotoxic lymphocytes.

(A-D) B16F10 cells were cultured overnight on substrates of differential rigidity, and stiffness measurements of individual cells performed by AFM (A-B) or SMR (C-D). A and C show schematic diagrams of the approach. Note that the higher throughput SMR approach enabled assessment of more substrate rigidities. (B, D) Cell stiffness measurements at the indicated substrate rigidities, determined by AFM (B) and SMR (D). Violins encompass the entire data distribution, with dashed lines denoting the median and dotted lines indicating the upper and lower quartiles. Samples sizes (n=) are displayed above (B) or below (D) each violin. P-values calculated by one-way ANOVA. (E-H) B16F10 cells cultured overnight on substrates of differential rigidity were mixed with OT-1 CTLs in the presence of OVA, followed by quantification of B16F10 killing and CTL cytokine production. Analogous studies were performed using NK cells as cytotoxic lymphocytes. (E) Schematic diagram of the approach. (F) CTL cytokine production, expressed as the percentage of TNF⁺IFNγ⁺ CTLs after 5 h coculture with B16F10 cells. (G) B16F10 killing by CTLs, measured by propidium iodide uptake into dead cells after 5 h in the presence or absence of OVA. (H) B16F10 killing, measured by propidium iodide uptake after 5 h in the presence or absence of NK cells. All results are representative of at least two independent experiments.

Fig. 2:. Preferential suppression of bone metastasis by cytotoxic lymphocytes.

Luc⁺ B16F10 cells were injected i.v. into wild type and Prf1^−/− mice, which were then monitored for metastatic colonization of the lungs and bone. (A) Schematic diagram of the experimental approach. (B) Representative IVIS images of tumor-bearing wild type and Prf1^−/− mice, with metastatic burden in the lungs and femurs indicated by black and yellow arrowheads. (C) Quantification of relative femoral colonization, expressed as a ratio of IVIS signal in the legs to the total IVIS signal. Error bars denote standard error of the mean (SEM). Sample size is indicated above each bar. P-value calculated by unpaired Student’s t-test. (D-E) Representative H&E images of B16F10 bone metastases in the femur (D) and spine (E) of a Prf1^−/− mouse. Epiphysis and metaphysis of the femur are indicated. Scale bars = 200 μm. Tumor cells indicated by black and yellow arrowheads. Scale bars = 200 μm. (F) Survival and paralysis-free survival of tumor-bearing wild type and Prf1^−/− mice. (G) H&E images of representative metastatic tumors from the indicated tissues are shown above, with immunofluorescence staining of the boxed regions shown below, with NKp46⁺ NK cells in red. WT = wild type. Black and yellow arrowheads indicate NK cell clusters in the Prf1^−/− bone. All results are representative of at least two independent experiments. Scale bars = 200 μm for H&E, 100 μm for immunofluorescence.

Fig. 3:. NK cells preferentially suppress metastatic outgrowth in rigid environments.

Luc⁺ B16F10 cells were injected i.v. into IgG control and NK depleted (NK^dep.) recipient mice, which were then monitored for metastatic colonization of the lungs and bone. (A) Schematic diagram of the experimental approach. (B) Representative IVIS images of tumor-bearing IgG control and NK^dep. mice, with metastatic burden in the lungs and femurs indicated by black and yellow arrowheads. (C) Quantification of relative femoral colonization, expressed as a ratio of IVIS signal in the legs to the total IVIS signal. Error bars denote SEM. Sample size is indicated above each bar. P-value calculated by unpaired Student’s t-test. (D) Survival and paralysis-free survival of tumor-bearing control and NK^dep. mice. (E) Representative H&E image of B16F10 bone metastasis in the spine of an NK^dep. mouse. Tumor cells indicated by black and yellow arrowheads. Scale bar = 500 μm. (F-H) C57BL/6 mice bearing stiff and soft subcutaneous hydrogel implants were treated with IgG control or NK cell depleting antibodies and then i.v. injected with Luc⁺ B16F10 cells. B16F10 colonization of the implants was assessed after 2 weeks. (F) Schematic diagram of the experimental approach. (G) Representative images of hydrogel implants three weeks after implantation, with black and yellow arrowheads denoting vascularization. Scale bars = 2.5 mm. (H) Implant colonization by B16F10 cells in IgG control (left) and NK^dep. (right) mice, measured by luciferase luminescence after hydrogel lysis. P-value calculated by paired Mann-Whitney test. All results are representative of at least two independent experiments.

Fig. 4:. Environmental and immune regulation of MTC stiffness and gene expression.

GFP⁺ B16F10 cells were i.v. injected either into wild type or Prf1^−/− mice or into IgG control treated or NK cell depleted (NK^dep.) mice. After 2 weeks, MTCs from the resulting metastases in the lungs of wild type and IgG control mice and the lungs and bones of Prf1^−/− mice and NK^dep. mice were extracted and subjected to SMR and scRNA-seq. (A) Schematic diagram of the experimental approach. (B-C) SMR of B16F10 MTCs isolated from the indicated organs of wild type and Prf1^−/− mice (B) or from IgG control and NK^dep. mice (C). Violins encompass the entire data distribution, with dashed lines denoting the median and dotted lines indicating the upper and lower quartiles. Samples sizes (n=) are displayed below each violin. P-values calculated by one-way ANOVA. (D) UMAP visualization of scRNA-seq data from the indicated lung and bone metastases. Cells are clustered based on transcriptional similarity and are color-coded by Seurat cluster identity. Cluster 7 has been boxed in each graph. (E) Pie charts showing the proportion of cluster 7 cells in each MTC sample. (F) Violin plot showing Opn expression levels across Seurat clusters in the indicated lung and bone metastases. Each violin represents the distribution of Opn expression within a cluster, with width indicating more cells. (G) qRT-PCR analysis of Opn expression levels in MTCs extracted from the indicated organs in IgG-treated versus NK^dep. tumor-bearing mice. Error bars denote SEM. P-values calculated by one-sample Wilcoxon test. All results are representative of at least two independent experiments.

Fig. 5:. Opn mediates cancer cell mechanoreciprocity and cell stiffening.

(A-C) The indicated Opn-KO B16F10 cell lines, along with nontargeting B16F10 control cells (NT), were subjected to AFM (A) and SMR (B) to measure their stiffness. (C-D) Phalloidin staining of Opn-KO1 and NT B16F10 cells. (C) Images of representative cells, with nuclei visualized by DAPI staining. Scale bars = 8 μm. (D) Quantification of F-actin intensity, with error bars indicating SEM. P-value calculated by unpaired Student’s t-test. (E) The indicated B16F10 cell lines were treated with purified Opn protein overnight and then subjected to SMR analysis. (F-G) The indicated B16F10 cells were loaded with increasing amounts of OVA and then mixed with OT-1 CTLs. (F) Target cell killing was measured by PI influx after 5 h. (G) CTL cytokine production, measured by intracellular staining for TNF and IFNγ, after 5 h. (H-I) NT or Opn-KO B16F10 cells were cultured overnight on substrates of differential rigidity and then subjected to SMR analysis. (H) Schematic diagram of the experimental approach. (I) Stiffness measurements at the indicated substrate rigidities. P = plastic. In A, B, E, and I, violins encompass the entire data distribution, with dashed lines denoting the median and dotted lines indicating the upper and lower quartiles. Samples sizes (n=) are displayed below each violin. P-values calculated by one-way ANOVA. All results are representative of at least two independent experiments.

Fig. 6:. Opn is required for mechanoreciprocity and mechanosurveillance in vivo.

(A-C) Luc⁺ NT or Opn-KO1 B16F10 cells were injected i.v. into wild type and Prf1^−/− mice, which were then monitored for metastatic colonization of the lungs and bone. (A) Schematic diagram of the experimental approach. (B) Representative IVIS images of tumor-bearing wild type and Prf1^−/− mice injected with the indicated B16F10 lines. (C) Quantification of relative femoral colonization, expressed as a ratio of IVIS signal in the legs to the total IVIS signal. (D-F) Luc⁺ NT or Opn-KO1 B16F10 cells were injected i.v. into IgG control and NK depleted (NK^dep.) recipient mice, which were then monitored for metastatic colonization of the lungs and bone. (D) Schematic diagram of the experimental approach. (E) Representative IVIS images of tumor-bearing IgG control and NK^dep. mice injected with the indicated B16F10 lines. (F) Quantification of relative femoral colonization, expressed as a ratio of IVIS signal in the legs to the total IVIS signal. In B and E, metastatic burden in the lungs and femurs is denoted by black and yellow arrowheads. In C and F, error bars denote SEM, sample size is indicated above each bar, and P-values were calculated by one-way ANOVA. (G-I) NT and Opn-KO2 B16F10 cells were subjected to comparative bulk RNA-seq. (G) Heat map showing downregulation of selected ECM, adhesion, and cytoskeleton-related genes in Opn KO2 cells. (H) Gene Set Enrichment Analysis (GSEA) showing downregulation of genes related to focal adhesions (left) and ECM (right). NES = normalized enrichment score. (I-J) GFP⁺ NT or Opn-KO B16F10 cells were injected i.v. into wild type and Prf1^−/− mice, and after 2 weeks, MTCs from the resulting lung metastases were extracted and subjected to SMR. (I) Schematic diagram of the experimental approach. (J) SMR of the indicated MTCs extracted from the indicated tumor-bearing mice. Violins encompass the entire data distribution, with dashed lines denoting the median and dotted lines indicating the upper and lower quartiles. Samples sizes (n=) are displayed below each violin. P-values calculated by one-way ANOVA. All results are representative of at least two independent experiments.

Fig. 7:. Cancer cell stiffness reflects environmental rigidity and immune pressure in human tumors.

(A-D) scRNA-seq analysis of cytoskeletal gene expression in breast cancer patients treated with ICB. (A) Schematic diagram illustrating the study design: Breast cancer biopsies from patients with the indicated disease subtypes were collected before and 9–11 days after a single dose of anti-PD-1 immunotherapy, then subjected to scRNA-seq. (B) UMAP visualization of scRNA-seq data colored by patient ID (left), outcome (top right), and treatment status (bottom right). (C) F-actin cytoskeleton gene expression in non-responder versus responder tumors, calculated using data from pre-treatment samples. Module scores were generated using the KEGG “Regulation of Actin Cytoskeleton” pathway and a GSEA “Cytoskeleton” gene set. Embedded boxes indicate median and interquartile range. P-values calculated by Wilcoxon rank-sum test. (D) Dot plot showing relative levels of selected highly expressed components of the F-actin, intermediate filament, and microtubule cytoskeletons. (E-G) Comparative analysis of MTCs from the osseous and epidural regions of spinal metastases. (E) Schematic diagram illustrating the anticipated differences in the immunosurveillance of breast cancer versus NSCLC metastases. (F-G) Left panels, SMR of epidural versus osseous MTCs isolated from the spine of patients with metastatic breast cancer (F) or NSCLC (G). Right panels, representative IHC images showing CD8 and CD56 staining in epidural breast cancer (F) or NSCLC (G) sections. Scale bars = 100 μm. In F and G, violins encompass the entire data distribution, with dashed lines denoting the median and dotted lines indicating the upper and lower quartiles. Samples sizes (n=) are displayed below each violin. P-values calculated by unpaired Student’s t-test.