Intrinsic self-defense pathways of tumor cells severely impair therapeutic potencies1,2,3, leading to frequent tumor recurrence and metastasis. For instance, heat shock proteins (HSPs) in tumor cells are stressfully upregulated to repair cell injury upon abnormal hyperthermia that can often be afforded by photothermal conversion agents such as copper sulfide (CuS)-based nanoparticles under light irradiation4,5,6,7, or non-thermal factors such as oxidants and free radicals8,9,10, while transforming growth factor β (TGFβ) pathway at tumor causes inaccessibility of antitumor therapeutics through elevated cascade proliferation and activation of cancer-associated fibroblasts (CAFs) to induce excessive enrichment of extracellular matrix (ECM) in tumors11,12, further severely compromising antitumor efficacy of conventional therapeutic compounds against fibrotic tumors such as pancreatic ductal adenocarcinoma (PDAC) tumors, together with inevitable tumor recurrence and metastasis11,13. Although relevant small-molecule inhibitors have been extensively explored to dismantle self-defenses of tumors for improving therapeutic potencies14,15,16, such compounds still suffer from severe dose-limiting off-target toxicities owing to their indiscriminate suppression of stressfully overexpressed proteins in tumor and normally expressed proteins in healthy tissues that are necessarily involved in crucial intracellular events such as HSPs-assisted protein folding correction and TGFβ-mediated tissue repair3,8. Hence, exploring a specific and efficient tool to selectively dismantle self-defenses of tumors is still urgently demanded for safely amplifying cancer therapy.

Transient receptor potential vanilloid member 1 (TRPV1) channel as a calcium-permeable channel, is involved in a diversity of pathophysiological processes (e.g. temperature and pain perception) and can be activated by multiple physicochemical stimuli such as heat (>42 °C), low pH, pungent chemicals and endogenous nociception mediators17,18,19. Moreover, TRPV1 channel is found to be overexpressed in a variety of aggressive tumors including breast, lung, hepatocellular, colorectal and pancreatic tumors, and is closely associated with in vitro proliferation, migration and survival of tumor cells20,21,22. Inspired by the observations that TRPV1 channel was relevant to the expressions of HSP70 and TGFβ proteins18,23,24, we hypothesize that TRPV1 channel might be involved in the modulation of self-defense behaviors of tumor cells during cancer therapy.

In this work, we show that nanoparticle-mediated TRPV1 blockade selectively suppresses stressful HSP70 and TGFβ1 via effective modulation of heat shock factor 1 (HSF1) for augmented thermo-immunotherapy against highly malignant tumors. Via applying the A549-TRPV1 knockdown (A549-TRPV1 KD) tumor model and transcriptome analysis, TRPV1 blockade is found to specifically block calcium influx upon hyperthermia at tumor, and results in distinct inhibition of HSF1 nuclear translocation for selectively suppressing stressfully overexpressed HSP70 to reverse thermo-resistance. Furthermore, tumor-selective TRPV1 blockade using polymeric micelles incorporating both indocyanine green (ICG) and TRPV1 antagonist yields considerable antitumor potency against a variety of primary tumors (e.g. breast, liver, colorectal, and pancreatic tumors), metastatic tumors, and recurrent tumors under light exposure, together with superior safety. More importantly, the inhibition of HSF1 nuclear translocation from this TRPV1 blockade distinctly attenuates TGFβ1 for effective decomposition of ECM to improve the infiltration of antitumor therapeutics (e.g. anti-PD-L1 antibody, aPD-L1) and immune cells into highly fibrotic and immunosuppressive tumors such as PDAC model, eventually achieving synergistic thermo-immunotherapy against both subcutaneous and orthotopic tumor models through the reinvigorated immune responses and alleviated immunosuppression. Such nanoparticles-mediated TRPV1 blockade provides an emerging paradigm to dismantle self-defenses of tumors for safely amplifying cancer therapy against highly intractable tumors.


TRPV1 blockade or knockdown enhances thermo-cytotoxicity

To generate potent hyperthermia for yielding cancer thermotherapy, the albumin nanoparticles caging copper sulfide nanocrystals (CuS-NCs) were constructed as a photothermal source as described previously25,26, which possessed the core size of 7.8 nm and hydrodynamic diameter of 25.4 nm (Supplementary Fig.1a). These CuS-NCs exhibited the concentration-dependent temperature elevation with the increase of30 °C during 300 s at the concentration of 1.0 mM Cu under near-infrared light irradiation, showing a distinct photothermal conversion capacity for causing hyperthermia owing to the notable near-infrared absorbance (Supplementary Fig.1b, c). To evaluate the influence of TRPV1 blockade on hyperthermia-mediated cytotoxicity from CuS-NCs, a specific TRPV1 antagonist SB70549827, was applied to wild-type A549 (A549-WT) cells that were simultaneously treated with CuS-NCs as well, followed by 5 min light exposure at 1.5 W cm−2and subsequent assessment of photocytotoxicity against A549-WT cells using MTT assay. The viability of A549-WT cells remained relatively unchanged after incubation with SB705498 or CuS-NCs without light exposure (Supplementary Fig.2a, b). However, upon hyperthermia from CuS-NCs (0.2 mM), SB705498 (0–20 nM) displayed the concentration-dependent improvement of cytotoxicity, and 40 nM SB705498 caused no obvious increase of cytotoxicity as compared to 20 nM SB705498 (Supplementary Fig.2c). In subsequent experiments, 20 nM SB705498 was used to block TRPV1 ion channels for potentiating thermo-cytotoxicity. In the presence of SB705498, the hyperthermia from CuS-NCs had the IC50of 0.25 mM under light exposure, whereas a distinct increase of IC50(0.39 mM) was observed in the absence of SB705498 under light exposure (Fig.1a). Meanwhile, SB705498 was also found to amplify hyperthermia-mediated cell injury via increasing the cell culturing temperature (Supplementary Fig.2d). These results suggest that the SB705498-mediated TRPV1 blockade accounts for the distinct improvement of thermo-cytotoxicity. Moreover, the 5-ethynyl-20-deoxyuridine (EdU) staining, which is frequently applied to detect proliferative cells during S phase through monitoring green fluorescence28, was performed to verify the ability of SB705498-mediated TRPV1 blockade to improve the thermo-cytotoxicity of CuS-NCs. Without light exposure, no matter combining SB705498 or not, CuS-NCs had no damage against A549-WT cell proliferation (Supplementary Fig.3). On the contrary, upon light exposure, SB705498 distinctly promoted the hyperthermia-based cytotoxicity as evidenced by the lowest green fluorescence intensity, revealing less proliferative cells during S phase (Supplementary Fig.3). Hence, the SB705498-mediated TRPV1 blockade dramatically improves the thermo-cytotoxicity upon hyperthermia.

Fig. 1: TRPV1 blockade or knockdown enhances thermotherapeutic efficacies via blocking Ca2+influx and inhibiting HSF1 nuclear translocation to suppress stressful HSP70 upregulation.
figure 1

aThermo-cytotoxicity of hyperthermia from CuS-NCs against A549-WT cells in the presence or absence of SB705498 (n= 3 biological replicates).bFluorescent images and TRPV1 expression of stably transfected A549-TRPV1 knockdown cells (A549-TRPV1 KD). Scale bars, 100 μm.cThermo-cytotoxicity of hyperthermia from CuS-NCs against A549-WT and A549-TRPV1 KD cells under light exposure (n= 3 biological replicates).dSchematic illustration of the synergistic mechanism of TRPV1 blockade with thermotherapy at tumor site. TRPV1 blockade effectively inhibits the calcium influx that induces heat shock factor 1 (HSF1) translocation into nucleus upon hyperthermia, leading to selective suppression of stressful HSP70 to dismantle the self-defense of tumor cells for preferable thermotherapeutic efficacy. Dash lines indicate the failure of downstream signals transduction. Ca2+influx imaging (red) (e), CLSM images of HSF1 (red) (f) and HSP70 (red) (g) in A549-WT and A549-TRPV1 KD cells treated with or without hyperthermia from CuS-NCs in the presence or absence of SB705498 or EGTA. Scale bars, 100 μm.hTemperature elevation at tumor site of mice bearing subcutaneous A549-WT and A549-TRPV1 KD tumors at 24 h post-injection of CuS-NCs under light exposure. Inset is infrared thermography of mice at 5 min during irradiation (Color bar, Low represents 15 °C and High represents 50 °C).iTumor volume of the mice bearing subcutaneous A549-WT and A549-TRPV1 KD tumors treated with hyperthermia from CuS-NCs together with intratumoral injection of SB705498 or not (n= 5 mice per group).jCLSM images of HSP70 (red) and TUNEL stainings (red) in the tumor sections from A549-WT or A549-TRPV1 KD tumor-bearing mice treated with hyperthermia from CuS-NCs together with intratumoral injection of SB705498 or not. Scale bars, 50 μm. Data are presented as mean ± SD (a, c, i). Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test. The experiments forb, e, f, g, andjwere repeated three times independently with similar results. Source data are provided as a Source Data file.

To verify the synergy of TRPV1 blockade with the hyperthermia, we established the stable A549-TRPV1 knockdown (A549-TRPV1 KD) cells using TRPV1 shRNA with a green fluorescent protein (GFP) tag. As shown in Fig.1band Supplementary Fig.4, more than 90% of the cells were GFP-positive, and the TRPV1 expression level in A549-TRPV1 KD cells was distinctly lower than that in A549-WT cells, confirming the effective knockdown of TRPV1. Then, the MTT assay showed that the hyperthermia from CuS-NCs had a 1.7-fold higher IC50value of 0.35 mM in A549-WT cells than that in A549-TRPV1 KD cells (0.20 mM) (Fig.1c). However, the overexpression of TRPV1 ion channels in A549 cells greatly decreased the thermo-cytotoxicity of CuS-NCs with the IC50value of 0.45 mM (Supplementary Fig.5a, b). These results suggest that the TRPV1 knockdown in tumor cells distinctly promotes the thermo-cytotoxicity. Since both A549-WT and A549-TRPV1 KD cells had similar cellular uptakes of CuS-NCs (Supplementary Fig.5c), the knockdown of TRPV1 channel in tumor cells is reasonably involved in potentiating the hyperthermia-mediated thermo-cytotoxicity.

TRPV1 blockade amplifies thermo-cytotoxicity via blocking Ca2+influx and suppressing subsequent HSF1 nuclear translocation-mediated stressful HSP70 upregulation

Since TRPV1 blockade efficiently synergizes thermotherapeutic efficiency, we further explored the mechanism of SB705498 as a TRPV1 antagonist to evade thermo-resistance. TRPV1 channel, as a temperature-sensitive calcium ion channel that dominates calcium influx, participates in intracellular signal transduction17,18. We thus hypothesized that Ca2+influx might play a vital role during thermotherapy, which is probably associated with HSF1 nuclear translocation and subsequent stressful HSP70 upregulation (Fig.1d)29,30,31. To clearly observe the influx of Ca2+after various treatments, a red fluorescent Ca2+-binding dye (CalciFluorTMRhod-4, AM) was used. The hyperthermia from CuS-NCs induced a sharp increase of intracellular Ca2+in A549-WT cells as indicated by the potently improved red fluorescence intensity (7.9-fold over that of PBS group), while TRPV1 blockade by SB705498 or TRPV1 knockdown was found to distinctly inhibit this intracellular Ca2+increase (Fig.1eand Supplementary Fig.6a). To further validate whether the Ca2+influx depends on extracellular Ca2+, we utilized an extracellular Ca2+chelator, ethylene glycol-bis-(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA), to scavenge extracellular Ca2+in the medium. Clearly, scavenging extracellular Ca2+led to negligible increase of red fluorescence, suggesting a specific dependence of Ca2+influx on extracellular Ca2+(Fig.1eand Supplementary Fig.6a). Moreover, the flow cytometric analysis of red fluorescence from intracellular Ca2+using CalciFluorTMRhod-4, AM probe further confirmed the ability of TRPV1 blockade or knockdown to suppress Ca2+influx (Supplementary Fig.6b, c). Hence, the hyperthermia sensitively activates TRPV1 channel-mediated Ca2+influx (Fig.1d), which is distinctly disadvantageous to thermo-cytotoxicity, while this Ca2+influx is also able to be effectively inhibited by TRPV1 blockade.

We further demonstrated the influence of Ca2+influx on the thermo-cytotoxicity from CuS-NCs using the MTT assay, in which non-toxic EGTA as calcium scavenger at the dose of 2.0 mM caused the IC50of ~0.24 mM under hyperthermia from CuS-NCs (Supplementary Fig.7), being preferable to the cytotoxicity (~0.35 mM) from the hyperthermia alone. Hence, scavenging extracellular Ca2+using EGTA displays a similar behavior to non-toxic SB705498 as indicated in Fig.1a. Afterwards, the EdU staining further revealed that the hyperthermia in the presence of EGTA resulted in a preferable inhibitory effect on cell proliferation as compared to hyperthermia alone under light exposure (Supplementary Fig.8), confirming that blocking Ca2+influx through TRPV1 channel is able to enhance the thermo-cytotoxicity.

Afterward, the flow cytometry was also utilized to evaluate the apoptosis of A549-WT cells under different treatments. The CuS-NCs-mediated hyperthermia alone caused the apoptosis level of 62.3%, while effective inhibition of Ca2+influx through TRPV1 antagonist SB705498 or calcium chelator EGTA led to an apparent increase of apoptosis (75.2%) under hyperthermia from CuS-NCs upon light exposure (Supplementary Fig.9), indicating the hyperthermia-activated Ca2+influx distinctly impair the thermotherapeutic efficiency. In addition, the western blot was also utilized to validate the apoptotic behavior, and the enhanced expression of cleaved caspase-3 was observed for A549-WT cells under CuS-NCs-mediated hyperthermia when combining with SB705498, EGTA, or TPRV1 knockdown as compared to that under hyperthermia alone (Supplementary Fig.10). Notably, TRPV1 blockade synergizes thermo-cytotoxicity and promotes tumor cells apoptosisviaefficiently blocking Ca2+influx.