Articial “Nano-Targeted Cells” for Bimodal Imaging-Guided Tumor Cocktail Therapy

22 Background: Single therapeutic modality always has its limitations in combating metastatic lesions 23 with complicacy. Although the emerging immunotherapy exhibits preliminary success, solid tumors 24 are usually immunosuppressive, leading to ineffective antitumor immune responses and 25 immunotherapeutic resistance. Rational combination of several therapeutic modalities may 26 potentially become a new therapeutic strategy to effectively combat cancer. 27 Results: Poly lactic-co-glycolic acid (PLGA) nanospheres were constructed with photothermal 28 transduction agents (PTAs)- Prussian blue (PB) encapsulated in the core and chemotherapeutic 29 docetaxel (DTX)/ immune adjuvant- imiquimod (R837) loaded in the shell. Tumor cell membranes 30 were further coated outside PLGA nanospheres (designated as “M@P-PDR”), which acted as 31 “Nano-targeted cells” to actively accumulate in tumor sites, which was guided/monitored by 32 photoacoustic (PA)/ magnetic resonance (MR) imaging. Upon laser irradiation, photothermal effects 33 were triggered. Combined with DTX, PTT induced in situ tumor eradication. Assisted by immune 34 adjuvant R837, the maturation of DCs were promoted. Besides, DTX polarized M2-phenotype 35 tumor-associated macrophages (TAMs) to M1-phenotype, relieving immunosuppressive TME. 36 Integrating the above processes, the infiltration of cytotoxic T lymphocytes (CTLs) increased. The 37 primary tumors and metastasis were significantly inhibited when treated with “Nano-targeted cells” 38 based cocktail therapy. 39 Conclusion: “Nano-targeted cells” based therapeutic cocktail therapy is a promising approach to 40 promote tumor regression and counter metastasis/ recurrence. This approach


47
as surgical resection, radiotherapy, chemotherapy, etc., may cause severe side-effects to normal 48 tissues, and some patients may suffer from recurrence and metastases [2][3][4]. Therefore, it is crucial 49 to develop effective therapeutic strategies to eradicate tumors. In addition to safety and high 50 selectivity, recurrence and metastasis should also be prevented. In the past decades, we have 51 witnessed preliminary efficacy of emerging hyperthermia therapy (HTT) against malignant tumors 52 [5]. As one of the paradigms of HTT, photothermal therapy (PTT) takes advantage of localized 53 photothermal transduction agents (PTAs) to convert light energy into heat and subsequently raises 54 the temperature of the tumor site, thereby inducing cancer cell death. Powered by nanotechnologies, 55 PTT offers unparalleled advantages, such as noninvasiveness and extremely low toxicity to normal 56 tissues. In addition, PTAs can often warrant both diagnostic and therapeutic functions [6,7]. 57 Although PTT can inhibit the growth of primary tumors to some extent, it also exposed certain 58 disadvantages, such as limited light penetration, which could result in inadequate tumor tissues 59 ablation. Moreover, only one therapeutic modality always has its limitations in combating metastatic 60 lesions with complicacy [8,9]. In fact, recurrences after hyperthermic ablation are common [10][11][12], 61 which is urgent to be addressed. 62

In Vitro Photothermal Performance and PA/MR Bimodal Imaging of M@P-PDR 163
To study the photothermal performance of M@P-PDR, the temperature changes of M@P-PDR 164 (with concentration at 1, 2, 3, 4 and 5 mg mL -1 ) after 808 nm laser irradiation were monitored by an 165 infrared thermal camera (Fotri226, Shanghai, China). The laser power intensity was set at 1.5 W 166 cm -2 , 5 min. Besides, 5 mg mL -1 of M@P-PDR nanospheres were irradiated with different power 167 intensities (0.75, 1.00, 1.25 and 1.50 W cm -2 ) for 5 min. In addition, the photothermal stability of were also measured. 181

Targeting Capability and Cytotoxicity Evaluation of M@P-PDR 182
To investigate the targeting capability of these M@P-PDR nanospheres, 4T1 cells were seeded 183 in confocal-specific dishes for 24 h to allow adhere, and then DiI-labeled M@P-PDR or P-PDR 184 nanospheres suspensions were added at an equivalent PLGA concentration of 50 μg mL -1 . After 185 various incubation times (0.5, 1, 2, 3, and 4 h), the nuclei were stained with DAPI for confocal laser 186 were seeded in the upper chambers, and the immature DCs were seeded in the lower plates. They 205 were randomly divided into six groups including (i) control group, (ii) M@P-PDR group, (iii) 206 M@P-PD + laser group, (iv) M@P-PR + laser group, (v) P-PDR + laser group, and (vi) M@P-PDR 207 + laser group, and received the corresponding treatment, respectively. Then the 4T1 cells in the 208 upper chambers were harvested and incubated with the lower plates for another 24 h. Finally, DCs 209 were collected and stained with anti-CD11c-FITC, anti-CD86-PE and anti-CD80-APC (eBioscience, 210 Thermo Science, USA) for flow cytometry analysis. Otherwise, the supernatant was assayed by the 211 ELISA kit for the detection of IL-6, IL-12 and TNF-α. 212

Animal Models 213
Female BALB/c mice (6 weeks old) were purchased from Enswell Biotechnology Ltd 214 (Chongqing, China). All experimental protocols in this study were approved by the Animal Ethics 215 Committee of the Second Affiliated Hospital of Chongqing Medical University. To inoculate the 216 4T1 breast cancer model, 4T1 cells (1.2 × 10 6 cells per mouse) suspended in RPMI-1640 medium 217 were subcutaneously injected into the fifth mammary fat pad on the left side. 218

In Vivo Biosafety and Biodistribution of M@P-PDR 219
To evaluate the in vivo biosafety of these M@P-PDR "Nano-targeted cells", healthy BALB/c 220 mice were intravenously administrated with M@P-PDR nanospheres suspension (3 mg mL -1 , 200 221 μL per mouse). Mice were sacrificed at 1 d, 3 d, 7 d, 15 d and 30 d (n = 5) post the injection, and 222 then blood samples were collected for hematology analysis and serum biochemical tests, 223 respectively. Major organs (heart, liver, spleen, lung, and kidney) were subjected to H&E staining. 224 Mice injected with saline were set as control. 225 To explore the biodistribution and in vivo targeting behavior of these "Nano-targeted cells", 226 tumor-bearing mice were randomly divided into two groups (n = 3), they were then injected with 227 DiR-labeled M@P-PDR or P-PDR nanospheres (equivalent PLGA concentration at 3 mg mL -1 , 200 228 µL), respectively. Then, these mice were subjected to an in vivo fluorescence imaging system at 229 various time intervals post above administration to record the DIR fluorescence imaging. In the 230 meantime, the corresponding fluorescence intensities were analyzed. Finally, animals were 231 sacrificed to harvest the major organs (heart, liver, spleen, lung, and kidney) and tumors for ex vivo 232 fluorescence evaluation.

In Vivo Photothermal Performance and Tumor Growth Inhibition Evaluation 244
To mimic distant tumors, after 6 days of primary tumor incubation (day-7), the equivalent 4T1 245 cells were subcutaneously injected into the right mammary fat pads at day -1. Then, all tumor 246 bearing-mice were randomly divided into eight groups (n = 5) including: (i) saline group (control), 247 (ii) M@P-PDR group, (iii)M@P-DR + laser group, (iv) M@P-P + laser group, (v) M@P-PR + laser 248 group, (vi) M@P-PD + laser group, (vii) P-PDR + laser group, and (viii) M@P-PDR + laser group. 249 The mice were intravenously injected with the corresponding nanospheres (equivalent PLGA 250 concentration at 3 mg mL -1 , 200 µL), respectively. After 8 h of the injection, the tumors were 251 irradiated by an 808 nm laser (1.5 W cm -2 , 10 min). The temperature changes in tumor areas were 252 recorded by a thermal camera. On the 3rd-day post-treatments, one mouse in each group was 253 sacrificed for primary tumor and major organs (heart, liver, spleen, lung, and kidney) dissection. 254 Then, H&E staining and examination were performed. Besides, the tumor tissues were further 255 stained with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and heat 256 shock 70 kDa protein (HSP70). On day 9, the distant tumors were collected for proliferating cell 257 nuclear antigen (PCNA) staining. To monitor tumor progression, the mice were photographed, and 258 the tumor volume changes were measured. During the treatment periods, the bodyweight of the 259 mice was also recorded. and cytokines including IL-6, IL-12, TNF-α and IL-10 were analyzed using ELISA kits according 271 to the manufacturer's protocols. In addition, immunofluorescence staining was further conducted to 272 investigate the infiltrating immune cells in tumor tissues. 273

Design, Synthesis and Characterization of M@P-PDR 280
M@P-PDR nanospheres, unique "Nano-targeted cells" with PB NPs encapsulated in the core, 281 DTX/R837 loaded in the shell, and cancer cell membranes coated on the surface, were constructed 282 for a homologous targeted cocktail therapy that concurrently integrates PTT, chemotherapy and 283 immunotherapy to achieve the optimum anti-tumor effect. These "Nano-targeted cells" actively 284 accumulated in tumor sites due to the homologous targeting capability, which was guided/monitored 285 by PA/MR bimodal imaging. Upon laser irradiation, photothermal effects were triggered. Combined obtained P-PDR nanospheres were further coated with cancer cell membranes to construct M@P-293 PDR "Nano-targeted cells" with homologous targeting capability (Fig. 1A). The SEM image 294 indicated that M@P-PDR displayed a uniform and spherical morphology (Fig. 1B). M@P-PDR 295 "Nano-targeted cells" showed a more obvious coating compared to P-PDR (Fig. 1C), which could 296 be ascribed to the coverage by cancer cell membranes. Sodium dodecyl sulfate polyacrylamide gel 297 electrophoresis (SDS-PAGE) was used to further analyze the protein composition of these M@P-298 PDR "Nano-targeted cells", and the results showed that M@P-PDR nanospheres had almost the 299 same protein composition as the original 4T1 cell membrane (Fig. 1D), which further demonstrated 300 the success of cell membrane coating. The zeta potential of the P-PDR and M@P-PDR nanospheres 301 were -24.2±0.85 mV and -17.0±1.40 mV, respectively (Fig. 1E), which could potentially prolong 302 the blood circulation as well as benefit other applications in the biological milieu [43]. Dynamic 303 light scattering (DLS) showed that the average hydrodynamic diameter of the nanospheres slightly 304 increased from 297 nm to 326.4 nm after the cell membrane coating (Fig. 1F). Compared to the 305 UV-Vis spectrum of M@P-DR, the spectrum of M@P-PDR suspension presented a characteristic 306 absorption band of PB at 700 nm ( Fig. 1G), indicating the successful loading of PB NPs in M@P-307 PDR. The loading efficacy of PB NPs, R837 and DTX were calculated to be 37.28%, 78.57% and 308 69.67%, respectively, according to the standard curves (Fig. S1A, S1B) and liquid-mass 309 spectrometry analysis (Fig. S1C, S1D). Such high loading capacities for these drugs demonstrated 310 that PLGA nanospheres held great potential as promising nanocarriers for drug delivery, which also 311 has been reported by many previous studies [44]. concentrations (1, 2, 3, 4 and 5 mg mL ˗1 ), respectively. Significant laser-power-dependent ( Fig. 2A, 318 2B) and concentration-dependent (Fig. 2C, 2D) photothermal effects were observed. Moreover, 319 excellent photothermal heating/cooling-cycling stability was also demonstrated (Fig. 2E). On this 320 ground, M@P-PDRs can be used as PTCAs for subsequent PTT. 321 The high sensitivity and high spatial resolution of PA imaging facilitate the visualization of 322 nanocarriers in vivo [46]. The multi-wavelength PA signal spectrum of M@P-PDR nanospheres 323 showed that 740 nm was the optimal wavelength for PA imaging (Fig. S2). As shown in Fig. 2F, 324 the PA signal intensities of M@P-PDR suspensions increased in a significant concentration-325 dependent manner. MR imaging performance was also investigated. As shown in Fig. 2G (inset), 326 the brightness of the T1-weighted MR images increased with the concentration of M@P-PDR 327 nanospheres, and the pseudo-colored T1-mapping images also showed the same tendency. The 328 relaxation rate (R1 value) was calculated to be 0.113 mM -1 s -1 by measuring the relaxation time (Fig.  329   2G). With the enhanced PA/MR dual-modal imaging capacity, the metabolic profiles of these M@P-330 PDR nanospheres at tumor sites can be visualized, providing guidance/monitoring for the 331 subsequent cocktail therapy. 332

Biocompatibility assay of M@P-PDR 333
As a prerequisite for any clinical development, the biocompatibility of M@P-PDRs was 334 investigated both in vitro and in vivo. First, the cytotoxicity of M@P-PDR and P-PDR nanospheres 335 toward 4T1 cells was evaluated using a standard CCK-8 assay. After 24 h of coincubation, both 336 concentration was lower than 400 μg mL -1 (Fig. S3). To further investigate the biocompatibility of 338 M@P-PDR, the in vivo acute and relatively long-term toxicity of M@P-PDR were evaluated in 339 healthy BALB/c mice. Routine blood tests and serum biochemical assay were performed on days 1, 340 3, 7, 15 and 30 after intravenous administration of M@P-PDR (Fig. S4A, S4B). Compared with the 341 reference range of hematology data, all indicators of the treated mice and the control group remained 342 at normal levels. In addition, the major organs (heart, liver, spleen, lung and kidney) were collected 343 for H&E staining (Fig. S4C), and negligible histomorphological or pathological changes were 344 observed. All these results strongly demonstrated that the ideal high biocompatibility of M@P-PDR 345 nanospheres as a multitasking therapeutic agent, providing great potential for their further clinical 346 translation. 347

In Vitro Homologous Targeting Capacity of M@P-PDR 348
The effective intracellular uptake of M@P-PDR nanospheres is the key to improve their 349 therapeutic efficacy. Functionalized by adhesion proteins of cancer cells on the surface, cancer cell 350 biomimetic nanoplatforms are expected to exhibit specific homologous targeting capacity[31, 34, 351 37]. Therefore, the targeting capacity of these M@P-PDR "Nano-targeted cells" was evaluated 352 using CLSM. As shown in (Fig. 3A, S5A, and S5B), 4T1 cells treated with M@P-PDR "Nano-353 targeted cells" exhibited stronger red fluorescence than that of P-PDR nanospheres, indicating that 354 the cancer cell membranes-coating promoted the intracellular uptake of nanocarriers. Moreover, the 355 red fluorescence enhanced with the extension of coincubation time. This phenomenon was further 356 confirmed by flow cytometry quantitative analyses (Fig. 3B). For instance, after 2 h of incubation, 357 the intracellular uptake rate of the "Nano-targeted cells"-treated group reached to 61.67%, while the 358 P-PDR nanospheres-treated group was only 12.54%. The above results indicated that the presence 359 of cancer cell membranes facilitated the intracellular uptake of nanocarriers thus exerting more 360 effective therapeutic effects. PDR nanospheres against 4T1 cell was evaluated next. According to the results of the CCK-8 assay 365 (Fig. 4A), the cell viability in M@P-PR + L group was 46.14 ± 5.62 %, showing the high efficacy 366 of PTT against tumor cells. The M@P-PDR + L group showed a lower cell viability (18.75 ± 6.21%), 367 probably because the released DTX had a certain killing effect on cancer cells. The cell viability of 368 the P-PDR + L group was 30.93 ± 2.11%, which was lower than that of the M@P-PDR + L group, 369 as the cancer cell membrane modification could have promoted more therapeutic agents to 370 accumulate into the tumor cells to mediate the therapeutic processes. The cell viabilities of the laser 371 only group, M@P-PDR only group and the M@P-DR + L group were 91.67 ± 6.08%, 91.26 ± 6.70% 372 and 90.43 ± 5.87%, respectively, which were not statistically different compared with that of the 373 control group (95.30 ± 7.30%). The cell damages were also analyzed by flow cytometry (Fig. 4C), 374 and the results were consistent with CCK-8 results. Furthermore, cells after various treatments were 375 also stained with CAM/PI to distinguish the live (green fluorescence) and dead (red fluorescence) 376  were seeded in the upper and lower chambers, respectively (Fig. 5A). The maturation efficacy of 402 DCs was measured by flow cytometry (Fig. 5B, 5C). A slight increase in DC maturation was 403 observed in the M@P-PDR-treated group, which was probably due to the inevitable release of a 404 small amount of R837 from these nanospheres. Compared to the M@P-PD + L group (without 405 R837), the level of DC maturation in the M@P-PDR + L group was greatly increased, which further 406 indicated the role of R837 in promoting the maturation of DCs. Relevant cytokines (TNF-α, IL-6 407 and IL-12) that would be released by mature DCs were measured by ELISA assays next. It was 408 found that the M@P-PDR group and the M@P-PR + L group showed higher secretion levels than 409 the M@P-PD + L group, which could be attributed to the pivotal role of R837. Compared to the 410 untargeted P-PDR + L group, DCs in M@P-PDR + L group secreted much more cytokines probably 411 due to homologous targeting capacity mediated by cancer cell membranes (Fig. 5D-F). 412

Biodistribution and In Vivo MR/PA Bimodal Imaging 413
To monitor the biodistribution and in vivo targeting behavior of these M@P-PDR "Nano-414 targeted cells", fluorescence imaging of tumor-bearing mice was performed. DiR-labeled M@P-415 PDR and P-PDR nanospheres were intravenously injected, respectively. In the M@P-PDR-treated 416 group, obvious fluorescence signals at the tumor sites were observed. The signals increased with 417 injection time and reached a peak at 8 h (Fig. 6A, 6B). The mean fluorescence intensity of the 418 tumors was 13.70 ± 1.35 × 10 3 , which was 2.49-fold higher than that of the P-PDR-treated group 419 (5.50 ± 0.54 × 10 3 ), which could be resulting from the homologous targeting capacity of cancer cell 420 membranes. More importantly, significant fluorescence signals were still clearly visible at 24 h post-421 injection, indicating long-term retention of these M@P-PDR "Nano-targeted cells". Afterward, 422 tumors and major organs (heart, liver, spleen, lung, kidney) were dissected for ex vivo fluorescence 423 imaging. The fluorescent signals of tumors in the M@P-PDR group were evidently stronger than 424 that of the P-PDR group (p < 0.05) (Fig. S6A, S6B). These results clearly indicated that the cancer 425 cell membrane-coated nanospheres were endowed with superior active targeting ability, showing 426 promising possibilities for in vivo precise imaging and effective treatment. 427 The aforementioned in vitro experiments have confirmed that M@P-PDR could act as contrast 428 agents to enhance both PA imaging and T1-weighted MR imaging. Therefore, bimodal PA and MR 429 imaging performance were further investigated in vivo. As expected, in the M@P-PDR group, the 430 PA signals within tumor regions gradually enhanced with prolonged time, and reached a peak at 8 431 h post-injection (0.479 ± 0.022) in comparison with those at pre-injection (0.089 ± 0.003) (Fig. 6C,  432   6E，S6C). At 24 h post-injection, the PA signal intensities (0.433 ± 0.022) slightly decreased due 433 to the gradual clearance of these nanospheres from tumor tissues. In contrast, in the P-PDR group 434 without homologous targeting, the PA signals were significantly weaker throughout the time course 435 of the observation. The T1-weighted MR imaging results showed that the tumors in the M@P-PDR 436 group were clearly demarcated from the surrounding normal tissues with clear anatomical structures. 437 In addition, obvious bright enhancements were observed at the tumor areas over time, reached 438 to a peak at 8 h post-injection, and were sustained for 24 h (Fig. 6D, 6F). PSII was used for 439 quantitative analysis of the T1-weighted MR imaging enhancement. Specifically, the average T1-440 weighted signal intensities in the M@P-PDR group increased by 159.632 ± 8.549% at 8 h post-441 injection, whereas only 76.784 ± 3.346% enhancement rate was observed in the P-PDR group. The 442 pseudo-color images also clearly showed the enhancements (Figure S6D). The trend of MR imaging 443 is consistent with that of PA imaging and the enrichment had been reflected by in vivo fluorescence 444 imaging. These results indicated that surface modification of cancer cell membranes on P-PDR 445 structures contributed to the efficient accumulation of nanocarriers in tumor sites. Additionally, the 446 excellent PA/MR bimodal imaging performance of these M@P-PDR "Nano-targeted cells" can 447 provide the therapeutic time window and guide the NIR laser irradiation, achieving more precise 448 therapy delivery. 449

In Vivo Photothermal Performance 451
After the enrichment of the PTCAs in tumor areas, the local temperature would rise under laser 452 irradiation. The tumors were exposed to laser irradiation 8 h after intravenous injection of 453 nanospheres, and the temperature changes were monitored by an infrared thermal imaging system. 454 As shown in Fig. 7B-D

, temperatures of tumors presented a feeble increase in the M@P-PDR and 455
M@P-DR + L groups compared to the control group in the absence of laser or PB NPs. Significant 456 temperature increase was observed in the groups with concurrent presence of laser irradiation and 457 the PB components, demonstrating excellent in vivo photothermal performance. The temperature in 458 the M@P-PDR + L group increased to 62.7 ℃, which was much higher that of the P-PDR + L group 459 (51.7 ℃) without homologous targeting capacity. Assisted by cancer cell membrane coating, PTCAs 460 could accumulate in tumors more efficiently to achieve more efficient and uniform localized 461 hyperthermia. 462

Immune Responses Evaluation 463
Encouraged by the activation of DCs in vitro, the in vivo immune responses were evaluated 464 next. The experimental design is shown in Fig. 7A. Tumors were inoculated at both the left and 465 right mammary fat pads of mice in chronological order, and set as primary tumor (1 st ) and artificial 466 mimicked metastasis (2 nd ), respectively. The mice were randomly divided into eight groups and 467 administered with different treatments. The day when the treatments designated was set as day 0. 468 To analyze the DCs maturation level in vivo, primary tumors (1 st ) (Fig. 8A, 8B), metastatic tumor 469 (2 nd ) (Fig. S7A, S7B) and lymph node (Fig. S7A, S7C) were collected to make single-cell 470 suspensions for flow cytometry assay on day 9. Similar to the in vitro results, the integration of the 471 R837 immune adjuvant endowed M@P-PDR "Nano-targeted cells" with a much stronger ability to 472 promote DC maturation, accompanied by increased cytokine secretion in vivo. In detail, the M@P-473 PDR + L group induced the highest level of DC maturation (66.56 ± 2.78%), which was significantly 474 higher than the M@P-PD + L group without R837 (22.81 ± 4.26%), M@P-PDR "Nano-targeted 475 cells" alone (32.65 ± 2.84%), and P-PDR + L group without homologous targeting capability (54.55 476 ± 1.96%). After PTT combined chemotherapy, tumor tissues were damaged and tumor cell 477 When the host immune status changes, the levels of cytokines in vivo will change 495 correspondingly. Here, the levels of IL-6, IL-12, TNF-α and IL-10 in the eight groups were 496 investigated by ELISA assay on day 9. As showed in Fig. 9A-C, the levels of these cytokines were 497 consistent with the change of the host immune status (DC maturation and polarization of TAMs) 498 discussed before. The groups integrated with DTX downregulated the production of IL-10 (Fig. 9D), 499 further demonstrating that DTX has an excellent ability to promote the polarization of M1-500 phenotype to M2-phenotype TAMs. 501 CD8 + T cells, namely CTLs, are essential for the anti-cancer immune response. To evaluate the 502 T cell response in vivo, the spleens of mice were collected on day 9 and T cells in the spleens were 503 analyzed using flow cytometry. The results (Fig. 9E, 9F) showed that the infiltration of CD8 + T 504 cells in the M@P-PDR+L group was 35.50 ± 0.96%, which was significantly higher than the control 505 group (17.33 ± 1.13%), the M@P-PDR group (23.54 ± 1.83%), the M@P-DR+L group (25.70 ± 506 1.57%), the M@P-P+L group (18.01 ± 1.77%), the M@P-PR+L group (27.64 ± 1.86%), the 507 M@P-PD+L group (22.99 ± 2.18%), and the P-PDR+L group (31.09±1.71%), indicating that the 508 processes (PTT, chemotherapy, DC maturation and polarization of TAMs) mediated by M@P-PDR 509 "Nano-targeted cells" triggered excellent anti-tumor immune responses. Consistently, 510 immunofluorescence images of the primary and metastatic tumors also revealed a substantial 511 infiltration of CD8 + T cells (Fig. S8B). 512

In Vivo Antitumor Therapy for Primary and Metastatic Tumors 513
Encouraged by the satisfactory immune response, the M@P-PDR-based cocktail therapy could 514 be a promising candidate to combat distant tumors. In the following study, we investigated whether 515 such a strong immune response initiated by M@P-PDR was available for long-term inhibition of 516 metastatic tumors. The therapeutic efficacy was evaluated by monitoring the growth of primary 517 tumors and distant tumors. Compared with the primary tumors (1 st ) in the control group, all other 518 treated groups exhibited certain inhibition effects on tumor growth (Fig. 10A, 10B, S9, S10A). In 519 detail, no significant difference has been found between M@P-PDR group and M@P-DR + L group, 520 whereas the limited tumor growth regression occurred as a result of the release of DTX and R837 521 in tumor sites. However, the photothermal effect of M@P-P greatly inhibited tumor progression, 522 with 3.05 folds growing in comparison to primary tumor volume. Particularly, the primary tumors 523 in the M@P-PDR + L group were remarkably inhibited, suggesting the excellent anti-tumor 524 efficiency of such cocktail therapy that concurrently integrates PTT, chemotherapy and 525 immunotherapy. As for the distant tumor growth in Fig. 10A,10C, S9, S10B, it was found that the 526 tumors in M@P-PDR + L group were also effectively inhibited, which could be ascribed to the 527 strong immune response resulted from R837-induced DCs maturation (immune activation) and 528 DTX mediated polarization of TAMs (relief of immunosuppression). To compare the therapeutic 529 effects more directly, the tumors in each group were collected at 3 d post-injection for TUNEL and 530 HSP70 staining (Fig. 10D). The results showed that in the presence of both PTCAs and laser 531 irradiation, the expression of HSP70 was higher than other groups, presenting obvious red 532 fluorescence. The M@P-PDR + L group showed a higher expression of HSP70 than P-PDR + L, proliferation. In addition to the pathological examination, the survival rates of mice in each group 538 was monitored until day 57 (Fig. S10D). The mice in M@P-PDR + L group survived without 539 obvious tumor recurrence. These results evidently confirmed that the powerful systemic immune 540 response of M@P-PDR-based cocktail therapy effectively inhibited the growth of distant tumors, 541 provided a new strategy for PTT/chemotherapy/immune therapy. The H&E staining of major organs 542 (Fig. S11) and the negligible body weight changes (Fig. S12) further demonstrated the satisfactory 543 biosafety of this synergistic therapeutic modality. 544 545

Conclusion 546
In summary, we rationally proposed a "Nano-targeted cells"-based cocktail therapy, where PTT 547 combined with chemotherapy and immunotherapy, creating a "doomsday storm" for tumors. These 548 as-synthesized "Nano-targeted cells" actively accumulate to the tumor sites due to the homologous 549 targeting capability, which can be guided by PA/MR bimodal imaging. Upon laser irradiation, PTT 550 will be triggered and TAAs will be subsequently released. The released TAAs, together with the 551 immune adjuvant R837, drive the maturation of DCs, secreting cytokines including TNF-α, IL-6, 552 and IL-12. Furthermore, chemotherapeutic drug DTX polarizes protumoral M2-phenotype 553 oncogenic TAMs to tumoricidal M1-phenotype oncogenic TAMs, relieving immunosuppressive 554 TME, accompanied by the decrease of IL-10. The above processes promote the infiltration of CTLs 555 for treating distant metastasis. The primary tumors and metastasis are significantly inhibited. "Nano-556 targeted cells"-based therapeutic cocktail therapy is a promising approach to promote tumor 557 regression and counter metastasis/ recurrence.

Consent for publication 581
Not applicable. 582

Availability of data and materials 583
All data analyzed during this study are included in this published article.

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