"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.

Keywords: dual nanomedicines; extracellular matrix; matrix metalloproteinase inhibitor; metastatic cancer; tumor microenvironment.

Scheme 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

Figure 1

Characterization of nanoparticles. Size distribution and TEM images of (a, c) MATT‐LTSLs and (b, d) PTX‐Ns. (e) in vitro release profile of CF‐LTSLs at 42 or 37°C. (f) in vitro release profile of PTX‐Ns or Taxol at 37°C (mean ± SD, n = 3). MATT‐LTSLs, marimastat‐loaded thermosensitive liposomes; PTX‐Ns, paclitaxel nanocrystals; TEM, transmission electron microscope

Figure 2

Antitumor efficacy in vitro. (a) The apoptosis rate of 4T1 cells was determined by Annexin V‐FITC/PI staining after 48‐hr incubation with PTX‐Ns or Taxol at a PTX concentration of 10 μg/ml at 37°C. The lower‐left, lower‐right, upper‐right, and upper left quadrants represented the viable, early apoptotic, late apoptotic, and dead cells, respectively. (b) Cell viability assessed by MTT after incubation with PTX‐Ns or Taxol for 48 hr at 37°C (mean ± SD, n = 5, ^* p < .05, ^** p < .01). (c) Cell migration evaluated by scratch wound‐healing assay after 20‐hr incubation with 10 μg/ml MATT at 37°C. (d) Cell invasion performed by a Matrigel® Transwell assay with a 24‐hr incubation with 10 μg/ml MATT at 37°C. Prior to incubation, MATT‐LTSLs were treated at 42°C for 1 hr to release the drug. The migrated cells were stained blue. (e) Quantification of the migration rate determined by optical density (OD) ratio at 570 nm (mean ± SD, n = 4, ^** p < .01). MATT‐LTSLs, marimastat‐loaded thermosensitive liposomes; MTT, 3‐(4,5)‐dimethylthiahiazo (‐z‐y1)‐3,5‐diphenyltetrazoliumromide; PTX‐Ns, paclitaxel nanocrystals

Figure 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. (a) Tumor volume change‐fold curves. (b) Tumor weight. The tumor was weighed on day 16 postinjection. (mean ± SD, n = 5, ^* p < .05, ^** p < .01). (c) Digital picture of tumors harvested on day 16 postinjection. HT, hyperthermia; LTSLs, lysolipid‐containing thermosensitive liposomes; MATT, marimastat; PTX‐Ns, paclitaxel nanocrystals

Figure 4

Inhibition of angiogenesis and lung metastasis in vivo. (a) Immunochemistry of CD31 staining for microvessels (brown) in tumor tissues collected from 4T1 tumor‐bearing Balb/C mice at Day 16 after treatment. (b) Quantitative analysis of microvascular density (MVD). MVD was quantified by five representative fields of cell nuclei under an optical microscope. (c) Digital images of lungs collected from 4T1 tumor‐bearing Balb/C mice at day 16 after treatment. The arrow indicated the tumor nodules on the lungs. (d) Quantitative analysis of tumor nodules on lungs by counting the numbers (mean ± SD, n = 3, ^* p < .05, ^** p < .01)

Figure 5

Inhibition of matrix metalloproteinase (MMPs) in vivo. (a) The activity of MMP‐2 and MMP‐9 was measured by gelatin zymography. Clear bands indicated gelatin degradation by MMPs and the activity of MMPs. (b) Quantitative analysis of MMP activity from zymograms using a computer analysis program. (c) Western blot analysis of MMP‐2 and MMP‐9 expression of tumors. Quantitative analysis of (d) MMP‐2 and (e) MMP‐9 expression. β‐actin was used as a loading control. Tumors used in these experiments were collected from 4T1 tumor‐bearing Balb/C mice at Day 16 after treatment (mean ± SD, n = 3, ^* p < .05, ^** p < .01)

Figure 6

Inhibition of extracellular matrix (ECM) degradation in vivo. The expression of collagen was analyzed by Masson trichrome staining. The collagen was stained blue. Laminin (LN), fibronectin (FN) were analyzed by immunohistochemical staining. The LN and FN proteins were stained brown. Tumor tissues for these experiments were collected from 4T1 tumor‐bearing mice on Day 16 post‐treatment