Maximized nanodrug-loaded mesenchymal stem cells by a dual drug-loaded mode for the systemic treatment of metastatic lung cancer

<p>Mesenchymal stem cells (MSCs), exhibiting tumor-tropic and migratory potential, can serve as cellular carriers to improve the effectiveness of anticancer agents. However, several challenges, such as the safety issue, the limited drug loading, the conservation of stemness and migration of MSCs, still remain in the MSC-based delivery system. In the present study, a novel nano-engineered MSC delivery system was established by loading doxorubicin (DOX)–polymer conjugates for the systemic treatment of pulmonary metastasis of breast cancer. For the first time, a dual drug-loaded mode, endocytosis and membrane-bound, was adopted to achieve the maximum amount of DOX conjugates in MSCs. The <i>in vitro</i> studies revealed the loaded MSCs possessed multifunctional properties, including preservation of the stemness and migration of MSCs, excellent stability of drug loading, acid sensitive drug release and obvious cytotoxicity against 4T1 cells. The <i>in vivo</i> studies confirmed that the loaded MSCs mainly located and long stayed in the lung where the foci of metastatic tumor situated. Importantly, loaded MSCs can significantly inhibit the tumor growth and prolong the life span of tumor-bearing mice in contrast with DOX and DOX-conjugate. The present loaded MSCs system suggested a promising strategy to solve several issues existed in cell-based delivery systems. Especially for the problem of low drug loading, the strategy, simultaneously loading nanodrug in cells’ internal and membrane, might be the most desirable method so far and could be developed as a generalizable manner for cell-mediated tumor-targeted therapy.</p

SCCA expression correlates to high grade breast cancer in the CDP progression TMA.

<p>The three CDP progression TMA case sets (designated as Case Sets 3, 5, and 7 by CDP) were probed for SCCA expression and scored. The grading information was provided by CDP in 291 accountable samples.</p

SCCA expression correlates to high grade breast carcinomas.

<p>The cumulative results of the SCCA positivity against the breast carcinoma grading from the CDP progression and prognostic TMAs are shown. SCCA is negative in the 124 normal or non-neoplastic cases of breast tissue. SCCA is positive in 1 of the 330 (0.3%) Grade I cases, 16 of the 638 (2.5%) Grade II cases, and in 37 of the 392 (9.4%) Grade III cases.</p

SCCA expression correlates to a decreased overall survival and recurrence-free survival.

<p>IHC was performed using the SCCA antibody FL-390 on the CDP prognostic TMAs. Kaplan-Meier survival curves for all patients (A–B) and only Grade II and Grade III patients (C–D) with SCCA-positive and negative tumors were compared for overall survival (A&C) and recurrence-free survival (B&D).</p

Validation of SCCA antibodies.

<p>293T cells were transfected with either vector alone, Flag-SCCA1, or Flag-SCCA2 plasmids. (A) Cells were subjected to immunoblot analysis using three SCCA antibodies: FL-390 for SCCA1/2, 8H11 for SCCA1, and 10C12 for SCCA2, as well as Flag antibody and β-tubulin antibody. (B) Cells were fixed and embedded in paraffin. IHC was performed with FL-390 and 10C12 antibodies. (C) IHC was performed on normal human tissue using the antibody Clone FL-390. Scale bars  = 50 µm.</p

Elevated SCCA expression is found in human breast cancer cell lines and human breast cancer carcinomas, but not in normal breast epithelium.

<p>(A) A panel of human cancer cells was probed for SCCA expression by immunoblotting. Five out of the six breast cancer cell lines (denoted with an asterisk) were positive for SCCA expression. (B) IHC analysis was performed on an array of breast carcinomas and normal breast tissue obtained from CHTN, using antibody FL-390. Representative images of normal breast tissue and sections with Grade III invasive ductal carcinoma are shown. (C) IHC staining was performed on the NCI CDP breast cancer progression and the prognostic TMAs using antibody FL-390. SCCA staining was scored on a tiered-scale (0–3). A representative panel is shown. Scale bars  = 50 µm.</p

SCCA expression correlates to advanced stage breast carcinomas.

<p>The Stage I, Stage II, and Stage III prognostic TMAs were obtained from CDP. IHC was against SCCA. The tissue was scored and the SCCA-positive cases are shown in parentheses against the number of cases for each stage. Note that all of the normal tissue were SCCA-negative, whereas 14 out of 573 (2.4%) Stage I, 12 out of the 391 (3.1%) Stage II, and 15 out of 174 (8.6%) Stage III cases were SCCA-positive.</p

SCCA expression correlates to high grade breast carcinoma in the CDP prognostic TMA.

<p>The Stage I (Case Sets 9–13), Stage II (Case Sets 14–17), and Stage III (Case Sets 18–19) prognostic TMAs were obtained from CDP, containing 598, 411, and 184 tissue specimens, respectively. IHC was performed against SCCA. The tissue was scored and the SCCA-positive cases are shown in parentheses against the number of cases in each grade. Note that all of the normal tissue were SCCA-negative, whereas 1 out of 294 Grade I, 11 out of the 534 Grade II, and 29 out of 310 Grade III cases were SCCA-positive.</p

Analysis of the Xyloglucan Endotransglucosylase/Hydrolase Gene Family during Apple Fruit Ripening and Softening

Ethylene and xyloglucan endotransglucosylase/hydrolase (<i>XTH</i>) genes were important for fruit ripening and softening in ‘Taishanzaoxia’ apple. In this study, we found it was <i>ACS1-1/-1</i> homozygotes in ‘Taishanzaoxia’ apple, which determined the higher transcription activity of <i>ACS1</i>. <i>XTH1</i>, <i>XTH3</i>, <i>XTH4</i>, <i>XTH5</i>, and <i>XTH9</i> were mainly involved in the early fruit softening independent of ethylene, while <i>XTH2</i>, <i>XTH6</i>, <i>XTH7</i>, <i>XTH8</i>, <i>XTH10</i>, and <i>XTH11</i> were predominantly involved in the late fruit softening dependent on ethylene. Overexpression of <i>XTH2</i> and <i>XTH10</i> in tomato resulted in the elevated expression of genes involved in ethylene biosynthesis (<i>ACS2</i>, <i>ACO1</i>), signal transduction (<i>ERF2</i>), and fruit softening (<i>XTHs</i>, <i>PG2A</i>, <i>Cel2</i>, and <i>TBG4</i>). In summary, the burst of ethylene in ‘Taishanzaoxia’ apple was predominantly determined by <i>ACS1-1/-1</i> genotype, and the differential expression of <i>XTH</i> genes dependent on and independent of ethylene played critical roles in the fruit ripening and softening. <i>XTH2</i> and <i>XTH10</i> may act as a signal switch in the feedback regulation of ethylene signaling and fruit softening

5′‑<i>C</i>‑Malonyl RNA: Small Interfering RNAs Modified with 5′-Monophosphate Bioisostere Demonstrate Gene Silencing Activity

5′-Phosphorylation is a critical step in the cascade of events that leads to loading of small interfering RNAs (siRNAs) into the RNA-induced silencing complex (RISC) to elicit gene silencing. 5′-Phosphorylation of exogenous siRNAs is generally accomplished by a cytosolic Clp1 kinase, and in most cases, the presence of a 5′-monophosphate on synthetic siRNAs is not a prerequisite for activity. Chemically introduced, metabolically stable 5′-phosphate mimics can lead to higher metabolic stability, increased RISC loading, and higher gene silencing activities of chemically modified siRNAs. In this study, we report the synthesis of 5′-<i>C</i>-malonyl RNA, a 5′-monophosphate bioisostere. A 5′-<i>C</i>-malonyl-modified nucleotide was incorporated at the 5′-terminus of chemically modified RNA oligonucleotides using solid-phase synthesis. <i>In vitro</i> silencing activity, <i>in vitro</i> metabolic stability, and <i>in vitro</i> RISC loading of 5′-<i>C</i>-malonyl siRNA was compared to corresponding 5′-phosphorylated and 5′-nonphosphorylated siRNAs. The 5′-<i>C</i>-malonyl siRNAs showed sustained or improved <i>in vitro</i> gene silencing and high levels of Ago2 loading and conferred dramatically improved metabolic stability to the antisense strand of the siRNA duplexes. <i>In silico</i> modeling studies indicate a favorable fit of the 5′-<i>C</i>-malonyl group within the 5′-phosphate binding pocket of human Ago2MID domain