“Locked” cancer cells are more sensitive to chemotherapy

Abstract The treatment of metastatic cancer is a great challenging issue throughout the world. Conventional chemotherapy can kill the cancer cells and, whereas, would exacerbate the metastasis and induce drug resistance. Here, a new combinatorial treatment strategy of metastatic cancer was probed via subsequentially dosing dual nanomedicines, marimastat‐loaded thermosensitive liposomes (MATT‐LTSLs) and paclitaxel nanocrystals (PTX‐Ns), via intravenous and intratumoral injection. First, the metastasis was blocked and cancer cells were locked in the tumor microenvironment (TME) by delivering the matrix metalloproteinase (MMP) inhibitor, MATT, to the tumor with LTSLs, downregulating the MMPs by threefold and reducing the degradation of the extracellular matrix. And then, the “locked” cancer cells were efficiently killed via intratumoral injection of the other cytotoxic nanomedicine, PTX‐Ns, along with no metastasis and 100% inhibition of tumor growth. This work highlights the importance of the TME's integrity in the chemotherapy duration. We believe this is a generalized strategy for cancer treatment and has potential guidance for the clinical administration.


| INTRODUCTION
Irrespective of the vast advance in cancer treatment, metastasis emerging in the entire stage of cancer progression is still a major cause of cancer-related death. 1,2 Particularly, breast cancer is a highly metastatic cancer and approximately 90% death of breast cancer patients was caused by the metastasis and resultant poor chemotherapy response. 3 Undeniably, preventing metastasis plays an essential role in the treatment of metastatic breast cancer. 4 Conventional chemotherapy can efficiently kill tumor cells, however, it facilitates the metastasis as well. 5 Metastasis is closely linked with the tumor microenvironment (TME) which consists of non-cancer cells, extracellular matrix (ECM), blood vessels, and lymphatics and is a sanctuary for cancer cells. 6,7 To kill the cancer cells efficiently, numerous formulations were designed to deliver the chemotherapeutic agents to the TME; nonetheless, the conventional formulation-based chemotherapy resulted in destruction of the TME induced by the killing of non-cancer cells like fibroblast and degradation of the ECM like collagen, [8][9][10][11] driving the cancer cells to escape from their broken "home (TME)" and exacerbating the metastasis. As a result, keeping the integrity of the TME would help inhibit the metastasis. The ECM-a major component of the TME, which is composed of a great number of macromolecules, such as proteins, glycoproteins, proteoglycans, and polysaccharides-is a critical scaffold of the TME and maintains its integrity. 12 However, the ECM would be discomposed by matrix metalloproteinases (MMPs) in the TME. 7 The MMPs are a type of zinc-dependent proteases secreted by cancer cells predominantly and can degrade the ECM components, such as collagens, gelatins, fibronectins, and other related proteins, leading to TME's destroy. 7,12,13 Accordingly, suppressing the MMPs is expected to keep the TME's integrity and then block the metastasis.
In our previous reports, we had demonstrated well that marimastat (MATT) was able to inhibit the MMPs and the resulted metastasis effectively via mimicking the substrate of the MMPs to work with MMPs in a competitive and reversible pattern. [14][15][16] MATT is of low toxicity toward cancer cells. However, its combined use with cytotoxicity agents, for example, paclitaxel (PTX) which is a potent antitumor drug acting via targeting the tubulin, can compensate the drawback and treat metastatic cancer. Previously by assembling prodrug polymer-PTX and MATT-loaded nanoparticles, coloaded nanocomplexes were designed to treat metastatic cancer in a 4T1 tumor-bearing model. 14,15 Although effective antitumor efficacy was demonstrated, the preparation of such complexes was too complicated to be characterized and to achieve the clinical transition.
Nanomedicines, including liposomes, [17][18][19] nanocrystals, 16,[20][21][22][23] biomimetic nanoparticles, [24][25][26][27][28] lipid drop-based nanocapsules, 29 polymer nanoparticles, [30][31][32][33][34][35] nanovaccines, 36,37 and so on, offer the promising potential to deliver the drug to the target site of interest with improved efficacy and reduced side effects. [38][39][40] Dual nanomedicinebased combinatorial therapy refers to coadministrating two separate nanomedicines. Compared with the single codelivery nanomedicinebased combined treatment, this strategy features advantages in terms of delivering two drugs to different action sites, flexible administration by diverse dose/time schedule/routes, avoiding drug-drug interaction and, in particular, ease scale-up and quality control, and being ready for regulation and commercialization as well, and so forth. [41][42][43] Intravenous administration of chemotherapeutic agent is a commonly used approach to treat cancer in the clinic; meanwhile, serious toxicity to the healthy organs is not avoided due to high drug level in the circulation. By contrast, the intratumoral injection has significantly higher local drug concentration at the tumor site that can efficiently induce the apoptosis of cancer cells and compromise the drug resistance. 44 Moreover, the local delivery decreases the systematic drug level dramatically and thus reduces the side effects 45. Here, based on the dual nanomedicines, MATT-loaded lysolipidcontaining thermosensitive liposomes (LTSLs) which the drug release was triggered by mild hyperthermia (HT) at 42 C and PTX nanocrystals (PTX-Ns) featuring extremely high drug-loading capacity, a new combinatorial treatment strategy of metastatic breast cancer was present. The two nanomedicines were administrated in a subsequential pattern. Unlike conventional combined-therapy strategy, MATT-LTSLs were intravenously injected, followed by HT treatment to stimulate MATT release in the TME to obtain an axial response-inhibit the expression of MMPs, suppress the ECM degradation, protect the TME integrity and "locked" the cancer cells in the TME. After that, the other cytotoxic nanomedicine, PTX-Ns, were intratumorally injected to kill the "locked" tumor cells, thus achieving tumor elimination without metastasis (Scheme 1).

| Nanoparticle preparation and characterization
LTSLs were prepared by a combined process of film hydration and ultrasonic treatment. The MATT-loaded LTSLs (MATT-LTSLs) had a particle size of 100 nm and a spherical morphology (Figure 1a,c), with S C H E M E 1 Design proposed an active mechanism for the sequential administration of MATT-LTSLs and PTX-Ns for targeting metastatic breast cancer.
Step 1: MATT-LTSLs are administrated via intravenous injection (i.v.). As MATT-LTSLs penetrate into the tumor tissue, MATT is released from the LTSLs triggered by local heating and diffused into a tumor site. The released MATT inhibits the activity of MMPs via associating with MMPs in the tumor microenvironment (TME) and, therefore, maintains the integrity of the TME and secondarily suppresses the migration of cancer cells.
Step 2: PTX-Ns are administrated via intratumor injection, followed by entering the tumor cells, releasing the PTX in the cytoplasm and killing the tumor cells. Compared with the traditional chemotherapy, injecting MATT-LTSLs in advance aims to inhibit the degradation of the extracellular matrix, block the tumor metastasis, and finally achieve tumor regression with high efficiency. MATT-LTSLs, marimastat-loaded thermosensitive liposomes; MMP, matrix metalloproteinase; PTX-Ns, paclitaxel nanocrystals encapsulation efficacy of approximately 60% assayed by highperformance liquid chromatography method. PTX-Ns were prepared using a precipitation-ultrasonication method using β-lactoglobulin To detect the thermosensitivity of LTSLs, a fluorescence probe (CF) was encapsulated in LTSLs and temperature triggered drug release was measured at physical temperature and 42 C. At physical temperature of 37 C, the release of CF was less than 30% at 2 hr. In contrast at

| Cellular uptake and cytotoxicity of PTX-Ns
Flow cytometry displayed that the cellular uptake of fluorescein isothiocyanate (FITC)-labeled PTX-Ns was concentration-and timerelated ( Figure S1 and Figure S2). Importantly, the intracellular fluorescence from the labeled nanoparticles was markedly greater than that of the free FITC, demonstrating an efficient uptake of the PTX-Ns.
Apoptotic study in 4T1 cells indicated that PTX-Ns allowed for apoptosis rate of 58%, greater than that from commercial formulation, Taxol, having a 51% rate (Figure 2a To examine the synergistic effects between MATT-LTSLs and PTX-Ns, we examined the cytotoxicity of the combined therapy ( Figure S3B) and calculated the combination index (CI) by fitting the curve of a coefficient of a drug interaction. As depicted in Figure S3C, the CI values of the combined therapy at the PTX/MATT (wt/wt) ratio of 2:1 were less than 1 when the inhibition rate (Fa) ranged from 0.3 to 0.9, therefore demonstrated the potential synergism between the two nanomedicine.   Figure S4F) and demonstrated that the LTSLs could penetrate inside the tumors and that the released payload in the nanoparticles had the potential to pass through the microvessels and locate in the TME.

| Inhibition of angiogenesis and lung metastasis
Angiogenesis is closely related with the metastasis. 14,15 Here, microvascular density (MVD) in the separated tumors collected at the end of treatment was investigated (Figure 4a,b). In the saline group, the MVD was 50 vessels per field. By contrast, the treatment with the single nanomedicine reduced the MVD by 2-3-fold and, more importantly, the MVD further declined by approximately twofold after the dual nanomedicine treatment.
The 4T1 tumor is a highly metastatic breast cancer, predominantly metastasizing to the lung and secondarily to the liver. 4,46 Here, the study of lung metastasis was performed by counting the nodules on the lung isolated at the end of treatment (Figure 4c,d). The control group treated with saline exhibited 15 nodules on the lung. By contrast, the treatment with MATT and, in particular, with MATT-LTSLs, reduced the number of tumor nodules to 6 and 2, respectively, being consistent well with the in vitro antimetastatic study described in  MMP-2 and MMP-9, also known as gelatinases, were highly expressed in breast cancer and play an essential role in metastasis and invasion. 7,12,13 In this study, the expression and activity of the two MMPs were evaluated by western blot assay and gelatin zymography, respectively, to probe the mechanism of antimetastasis. The activity F I G U R E 3 Antitumor efficacy in vivo in 4T1 tumor-bearing Balb/C mice. Free PTX, MATT, and the same volume of saline were injected via the tail vein every 3 days at the dose of 10 mg/kg for PTX and 5 mg/kg for MATT, respectively. HT treatment was performed immediately by placing the tumor inside a water bath at 42 C for 45 min after the animals were injected with 10% chloral hydrate (wt/vol) at a dose of 400 mg/kg. For the group treated with dual nanomedicines, the mice were administered with MATT-LTSLs in advance and, at 4 hr after HT treatment, were subjected to intratumor injection of PTX-Ns.  (Figure 5a,b); however, the maximal reduction was observed in the dual nanomedicine-treated group, with 1.5-and 1-fold decrease for MMP-9 and MMP-2, respectively. Western blot assay and quantified analysis indicated the expression of MMPs was decreased by 2.5-fold and 4-fold for MMP-2 and MMP-9, respectively, after treatment with the dual nanomedicines compared with the control saline ( Figure 5c-e). Indeed, besides the dual nanomedicines, the MATT-LTSLs displayed significant downregulation of the MMPs as well, demonstrating the potent delivery of MATT using LTSLs. In short, the dual nanomedicine-based approach allowed for profound inhibition of MMPs' activity and expression. Also, compared with using MATT-LTSLs or PTX-Ns alone, the dual nanomedicine-treatment could suppress the MMPs with higher efficacy and, as a result, demonstrated a potential synergistic effect between the two nanomedicines.

| Inhibition of ECM degradation
The ECM is a supportive scaffold of the TME and is critical to maintaining the integrity of TME. However, most of the ECM, such as collagen, laminin (LN), and fibronectin (FN), is MMPs's substrate 7,12 ; and accordingly, the highly expressed MMPs in tumor would discompose the ECM and thus jeopardize the TME's integrity. The experimental results present in Figure 5  was increased as well after treatment with MATT formulations. Compared with free MATT, the MATT-LTSLs allowed for higher expression of the three ECMs. Overall, these results demonstrated that the administration of MATT-LTSLs was able to suppress the degradation of ECM and thereby benefit the maintenance of the TME's integrity.

| DISCUSSION
MATT is able to inhibit the metastasis, but its efficient delivery to the tumor site is critical. MATT was used in clinical study for tumor therapy 20 years ago; however, it failed in the Phase III study due to the modest efficacy and the cumulative toxicity of inflammation and musculoskeletal pain. 47,48 After that, the exploitation of the drug was terminated. Surprisingly, recent studies unveiled that the drug could inhibit the metastasis, 14,15 alleviate the inflammation, 49 and regulate the immune system 50 via inhibition of MMPs. Indeed, our previous reports indicated the drug blocked the metastasis significantly. [14][15][16] On the other side, we found that the inhibition efficacy in metastasis from nanoparticle formulation was markedly different from that of the free drug, despite that their discrepancy of MMP inhibition in vivo was less than 20% ( Figure 5). 16 In this study, MATT-LTSLs enabled threefold of metastasic nodules less than the free MATT (Figure 4c,d), being in line with our previous reports. 14-16 Therefore, we speculate that abundant MMPs exist in the tumor site; and as a result, effective delivery of MATT to the tumor site with carriers is indispensable. The use of nanoparticles is possible to overcome the drawbacks of MATT but additional work is needed.
The "locked" cancer cells were more sensitive to chemotherapy.
Undeniably, chemotherapy is the first choice for patient with cancer for its extremely low cost. Nevertheless, the chemotherapy is a doubleedged sword that can kill the cancer cells and, meanwhile, would induce metastasis and drug resistance, rendering most of the patient ending with chemotherapy-response failure and death 51 ; and also, the metastasis which occurs in the entire stage of cancer development would be further exacerbated by the broken TME by chemotherapy. 11,52,53 Here, we uncovered poor response of 4T1 cells to the single treatment with free PTX or PTX-Ns. In contrast, the administration of MATT formulation followed by PTX-Ns displayed significant enhancement in apoptosis and inhibition of proliferation and tumor growth (Figure 3 and Figure S4). Because of the staying of TME's integrity by MATT, the metastasis was markedly blocked, an indicator that the cancer cells were fixed well in the TME (Figures 4 and 6). These results demonstrated that steady-state cancer cells are more sensitive to chemotherapy. This finding can provide a guide for cancer therapy in the clinic. The 4T1 cancer model is highly metastatic cancer. Subsequentially dosing the dual nanomedicines, MATT-LTSLs and PTX-Ns, via different routes could treat metastatic cancer with high efficacy. The two nanomedicines feature easy preparation, high payloads, and wellknown tumor-targeting ability, therefore allowing the strategy for ready clinical translation. We believe this is a universal strategy that can be adapted to treat other metastatic diseases as well. Although PTX-Ns-induced apoptosis with higher efficacy compared with free PTX, however, this nanomedicine exhibited lower cytotoxicity at high concentrations. Our previous reports also displayed similar results. [54][55][56] Furthermore, combined use with other drugs, such as gene and protein, made this enhancement more profound. 54,55 The previous report indicated that internalized PTX-Ns started to dissolve at 10 hr postinternalization, and therefore demonstrating sustained PTX release over time in cells. 57 Consequently, the sustained release might help the chemotherapeutic agent loaded in nanomedicines kill the cancer cells. Indeed, additional experiments are needed to study the potential mechanism.

| CONCLUSION
In summary, we have demonstrated a generalized strategy based on dual nanomedicines administered via a subsequential pattern for the targeted combination treatment of highly metastatic cancer such as breast cancer. Via first targeting delivery of the MMP inhibitor, MATT, to the tumor with LTSLs and the resultant maintenance of the TME's integrity, the metastasis is blocked and cancer cells are locked in the TME; and subsequently, the other cytotoxic nanomedicine, PTX-Ns, is intratumorally injected and kill the "locked" cancer cells efficiently, exhibiting as zero metastasis to the lung and almost 100% inhibition of tumor growth. This work highlights the potential importance of keeping the TME's integrity in the chemotherapy duration. Additionally, owing to the facile preparation of the nanomedicines with high drug-loading capacity, well-known biocompatibility and the readily dosing, the strategy present here has promising potential for clinical transition and can be applied to treating other metastatic diseases as well.

| Cell cultures
4T1 cells were cultured in RPMI 1640 medium containing 10% FBS and 1% penicillin streptomycin solution, and incubated at 37 C under 5% CO 2 . The cells were harvested with trypsin and cell suspensions were prepared for further experiments.

| Nanoparticle preparation and characterization
MATT-LTSLs were prepared as described in our previous report. 15 Briefly  The drug release from Taxol and PTX-Ns was tested by a dialysis method under a shaker at 37 C as well. The detailed process and PTX assay were described in our previous report. 54 5.5 | Flow cytometry 4T1 cells (1 × 10 5 ) seeded in 12-well plates for 48 hr were incubated with dye-labeled nanoparticles at 37 C. After that, the cells were harvested, washed with cold PBS and resuspended in 500 μl of PBS for flow cytometry analysis (Accuri C6, BD).

| Confocal imaging
Cells (2 × 10 5 ) seeded on 12 mm round glass coverslip in advance for cell attachment were incubated with dye-labeled nanoparticles in serum-free medium for 2 hr at 37 C, washed with cold PBS three times, reacted with 4% paraformaldehyde and stained with DAPI for 10 min, and finally observed with a LSM700 confocal laser scanning microscopy (CLSM, Carl Zeiss, Germany). And for combination therapy, the cells were first treated with MATT formulations for 4 hr and, then, incubated with PTX formulations for another 48 hr. The ratio of MATT and PTX was 1:2 (wt/wt). MATT-LTSLs were heated at 42 C in a water bath for 45 min before treatment with cells.

| In vitro cytotoxicity
After incubation, cells were cultured with MTT (1 mg/ml) for 4 hr, followed by the removing of culture medium, the addition of 150 μl DMSO and absorbance measurement at the wavelength of 570 nm using a microplate reader (Multiskan FC, Thermo Fisher Scientific). Additionally, based on the cytotoxicity, the coefficient drug interaction between the two nanomedicines was analyzed by the CompuSyn software.
The cell apoptosis was treated by Annexin V-FITC/PI apoptosis detection kits followed standard protocol and detected by flow cytometry (Accuri C6, BD).

| Wound healing test and transwell
4T1 cells (1 × 10 5 ) were seeded in 12-well plates for a cover density of 70-80% reached, followed by scratch making using a 200-μl pipette. After that, cells were cultured in a serum-free medium and treated with MATT formulations at a MATT concentration of 5 μg/ml. MATT-LTSLs were preheated at 42 C in a water bath for 1 hr. Images were taken at 0 and 20 hr postscratching with an optical microscope (Olympus IX53, Japan).
Transwell assays were performed in 24-well Transwell chambers with an 8-μm pore (Corning Life Sciences, Inc.), as described in our reports. 15,55 In brief, 4T1 cells seeded at a density of 1 × 10 5 cells per well in the upper chamber for 24 hr, were incubated with 200 μl of serum-free medium and treated with MATT formulations at a MATT concentration of 1 μg/ml for 4 hr at 37 C. MATT-LTSLs were preheated at 42 C in a water-bath for 1 hr before incubation with cells. Subsequently, the cells in the upper chamber were removed with a cotton swab and, meanwhile, the cells attached on the lower surface of the filter were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet for 10 min and washed. The stained cells were imaged by optical microscopy (Olympus IX53, Japan). Crystal violet was dissolved in 33% acetic acid and the optical density ratio was measured at the wavelength of 570 nm using a microplate reader (Multiskan FC, Thermo Fisher Scientific).

| Statistical analysis
Data were presented as means ± SD one-way analysis of variance was performed to assess the statistical significance of the differences between samples. p < .05 was considered significant.