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Induction of apoptosis of metastatic mammary carcinoma cells in vivo by disruption of tumor cell surface CD44 function.

Yu Q, Toole BP, Stamenkovic I - J. Exp. Med. (1997)

Bottom Line: To understand how the hyaluronan receptor CD44 regulates tumor metastasis, the murine mammary carcinoma TA3/St, which constitutively expresses cell surface CD44, was transfected with cDNAs encoding soluble isoforms of CD44 and the transfectants (TA3sCD44) were compared with parental cells (transfected with expression vector only) for growth in vivo and in vitro.However, although parental cells were dividing and forming clusters within lung tissue 48 h following injection, >80% of TA3sCD44 cells underwent apoptosis.Although sCD44 transfectants displayed a marked reduction in their ability to internalize and degrade hyaluronan, they elicited abundant local hyaluronan production within invaded lung tissue, comparable to that induced by parental cells.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathology Unit and MGH Cancer Center, Massachusetts General Hospital, Charlestown Navy Yard, Boston, Massachusetts 02129, USA.

ABSTRACT
To understand how the hyaluronan receptor CD44 regulates tumor metastasis, the murine mammary carcinoma TA3/St, which constitutively expresses cell surface CD44, was transfected with cDNAs encoding soluble isoforms of CD44 and the transfectants (TA3sCD44) were compared with parental cells (transfected with expression vector only) for growth in vivo and in vitro. Local release of soluble CD44 by the transfectants inhibited the ability of endogenous cell surface CD44 to bind and internalize hyaluronan and to mediate TA3 cell invasion of hyaluronan-producing cell monolayers. Mice intravenously injected with parental TA3/St cells developed massive pulmonary metastases within 21-28 d, whereas animals injected with TA3sCD44 cells developed few or no tumors. Tracing of labeled parental and transfectant tumor cells revealed that both cell types initially adhered to pulmonary endothelium and penetrated the interstitial stroma. However, although parental cells were dividing and forming clusters within lung tissue 48 h following injection, >80% of TA3sCD44 cells underwent apoptosis. Although sCD44 transfectants displayed a marked reduction in their ability to internalize and degrade hyaluronan, they elicited abundant local hyaluronan production within invaded lung tissue, comparable to that induced by parental cells. These observations provide direct evidence that cell surface CD44 function promotes tumor cell survival in invaded tissue and that its suppression can induce apoptosis of the invading tumor cells, possibly as a result of impairing their ability to penetrate the host tissue hyaluronan barrier.

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(A) Soluble CD44 inhibits events required for TA3/St cell growth in the lung tissue microenvironment. TA3neo No. 1 (a, c, and e) and  TA3sCD44v6-10 No. 17 (b, d, and f) cells were labeled with green CMFDA and 5 × 106 cells in 0.2 ml HBSS buffer were injected into the tail vein of  A/jax mice. The animals were killed at 1, 24, and 48 h after injection, the lungs were fixed and paraffin-embedded, and 5-μm-thick paraffin sections were  mounted onto slides and examined by fluorescence microscopy. 1 h after injection, both TA3neo No. 1 and TA3sCD44 v6-v10 No. 17 cells were observed to be arrested in pulmonary blood vessels (a and b) and to penetrate the pulmonary interstitium (c and d). Occasional cells appeared to be in the  process of extravasation (c, arrowhead and the arrow indicate an extravasating cell and the lumen of a blood vessel, respectively). 48 h after injection (e and  f), TA3 neo cells display cluster formation (e), whereas only a few isolated TA3sCD44v6-10 No. 17 cells remain in the lung parenchyma (f). Bar in a  and b = 304 μm; bar in c–f = 76 μm. (B) In situ detection of apoptosis by TUNEL assay. Lung sections showing TA3neo No. 1 cells (a and c) and  TA3sCD44v6-10 No. 17 cells (b and d) labeled with CMFDA (green fluorescence) or detected with ApopTag (red fluorescence) 1 h (a and b) and 48 h (c and  d) after intravenous injection. 1 h after injection, TA3neo No. 1 and TA3sCD44 v6-v10 No. 17 cells display similar distribution and no reactivity with  ApopTAG (red fluorescence). 48 h after injection, the majority of TA3neo cells were ApopTAG-negative (c) whereas most (>80%) of the remaining  TA3sCD44v6-v10 No. 17 cells tested positive for ApopTAG staining (d, arrows). Bar in a–d = 125 μm.
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Figure 3: (A) Soluble CD44 inhibits events required for TA3/St cell growth in the lung tissue microenvironment. TA3neo No. 1 (a, c, and e) and TA3sCD44v6-10 No. 17 (b, d, and f) cells were labeled with green CMFDA and 5 × 106 cells in 0.2 ml HBSS buffer were injected into the tail vein of A/jax mice. The animals were killed at 1, 24, and 48 h after injection, the lungs were fixed and paraffin-embedded, and 5-μm-thick paraffin sections were mounted onto slides and examined by fluorescence microscopy. 1 h after injection, both TA3neo No. 1 and TA3sCD44 v6-v10 No. 17 cells were observed to be arrested in pulmonary blood vessels (a and b) and to penetrate the pulmonary interstitium (c and d). Occasional cells appeared to be in the process of extravasation (c, arrowhead and the arrow indicate an extravasating cell and the lumen of a blood vessel, respectively). 48 h after injection (e and f), TA3 neo cells display cluster formation (e), whereas only a few isolated TA3sCD44v6-10 No. 17 cells remain in the lung parenchyma (f). Bar in a and b = 304 μm; bar in c–f = 76 μm. (B) In situ detection of apoptosis by TUNEL assay. Lung sections showing TA3neo No. 1 cells (a and c) and TA3sCD44v6-10 No. 17 cells (b and d) labeled with CMFDA (green fluorescence) or detected with ApopTag (red fluorescence) 1 h (a and b) and 48 h (c and d) after intravenous injection. 1 h after injection, TA3neo No. 1 and TA3sCD44 v6-v10 No. 17 cells display similar distribution and no reactivity with ApopTAG (red fluorescence). 48 h after injection, the majority of TA3neo cells were ApopTAG-negative (c) whereas most (>80%) of the remaining TA3sCD44v6-v10 No. 17 cells tested positive for ApopTAG staining (d, arrows). Bar in a–d = 125 μm.

Mentions: To address the fate of TA3sCD44 cells in vivo, and thereby gain insight into the stage of the metastatic process at which cell surface CD44 might be required, we labeled tumor cells with green CMFDA, which allows cell tracing for at least 48 h after injection. Animals injected with the labeled transfectants were killed at 1, 24, and 48 h after injection and lung tissue sections were examined by fluorescence microscopy. Both TA3neo and TA3sCD44 transfectants were found to arrest within lung blood vessels and extravasate into the interstitial stroma within 1 h after injection (Fig. 3 A). Between 1 and 24 h after injection, both TA3neo No. 1 and TA3sCD44v6-10 No. 17 cells were observed to invade lung stroma (Fig. 3 A, a–d, and B, a and b, and data not shown). However, at 48 h, when TA3neo transfectants were forming clusters, only isolated TA3sCD44v6-10 cells were visible (Fig. 3 A, e and f). To determine whether TA3sCD44v6-10 transfectants were undergoing apoptosis within the lung interstitium, TUNEL assays were performed on the tissue sections and TUNEL-positive cells were scored among 100 CMFDA-labeled cells. At 1 and 24 h after injection, minimal TUNEL staining was observed among TA3neo and TA3sCD44v6-10 cells alike (Fig. 3 B, a and b, and data not shown). However, at 48 h, 93 and 88% of the CMFDA-labeled TA3sCD44 cells tested positive for the TUNEL assay in contrast to 10 and 19% of the TA3neo transfectants in two independent experiments (Fig. 3 B, c and d, and data not shown).


Induction of apoptosis of metastatic mammary carcinoma cells in vivo by disruption of tumor cell surface CD44 function.

Yu Q, Toole BP, Stamenkovic I - J. Exp. Med. (1997)

(A) Soluble CD44 inhibits events required for TA3/St cell growth in the lung tissue microenvironment. TA3neo No. 1 (a, c, and e) and  TA3sCD44v6-10 No. 17 (b, d, and f) cells were labeled with green CMFDA and 5 × 106 cells in 0.2 ml HBSS buffer were injected into the tail vein of  A/jax mice. The animals were killed at 1, 24, and 48 h after injection, the lungs were fixed and paraffin-embedded, and 5-μm-thick paraffin sections were  mounted onto slides and examined by fluorescence microscopy. 1 h after injection, both TA3neo No. 1 and TA3sCD44 v6-v10 No. 17 cells were observed to be arrested in pulmonary blood vessels (a and b) and to penetrate the pulmonary interstitium (c and d). Occasional cells appeared to be in the  process of extravasation (c, arrowhead and the arrow indicate an extravasating cell and the lumen of a blood vessel, respectively). 48 h after injection (e and  f), TA3 neo cells display cluster formation (e), whereas only a few isolated TA3sCD44v6-10 No. 17 cells remain in the lung parenchyma (f). Bar in a  and b = 304 μm; bar in c–f = 76 μm. (B) In situ detection of apoptosis by TUNEL assay. Lung sections showing TA3neo No. 1 cells (a and c) and  TA3sCD44v6-10 No. 17 cells (b and d) labeled with CMFDA (green fluorescence) or detected with ApopTag (red fluorescence) 1 h (a and b) and 48 h (c and  d) after intravenous injection. 1 h after injection, TA3neo No. 1 and TA3sCD44 v6-v10 No. 17 cells display similar distribution and no reactivity with  ApopTAG (red fluorescence). 48 h after injection, the majority of TA3neo cells were ApopTAG-negative (c) whereas most (>80%) of the remaining  TA3sCD44v6-v10 No. 17 cells tested positive for ApopTAG staining (d, arrows). Bar in a–d = 125 μm.
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Figure 3: (A) Soluble CD44 inhibits events required for TA3/St cell growth in the lung tissue microenvironment. TA3neo No. 1 (a, c, and e) and TA3sCD44v6-10 No. 17 (b, d, and f) cells were labeled with green CMFDA and 5 × 106 cells in 0.2 ml HBSS buffer were injected into the tail vein of A/jax mice. The animals were killed at 1, 24, and 48 h after injection, the lungs were fixed and paraffin-embedded, and 5-μm-thick paraffin sections were mounted onto slides and examined by fluorescence microscopy. 1 h after injection, both TA3neo No. 1 and TA3sCD44 v6-v10 No. 17 cells were observed to be arrested in pulmonary blood vessels (a and b) and to penetrate the pulmonary interstitium (c and d). Occasional cells appeared to be in the process of extravasation (c, arrowhead and the arrow indicate an extravasating cell and the lumen of a blood vessel, respectively). 48 h after injection (e and f), TA3 neo cells display cluster formation (e), whereas only a few isolated TA3sCD44v6-10 No. 17 cells remain in the lung parenchyma (f). Bar in a and b = 304 μm; bar in c–f = 76 μm. (B) In situ detection of apoptosis by TUNEL assay. Lung sections showing TA3neo No. 1 cells (a and c) and TA3sCD44v6-10 No. 17 cells (b and d) labeled with CMFDA (green fluorescence) or detected with ApopTag (red fluorescence) 1 h (a and b) and 48 h (c and d) after intravenous injection. 1 h after injection, TA3neo No. 1 and TA3sCD44 v6-v10 No. 17 cells display similar distribution and no reactivity with ApopTAG (red fluorescence). 48 h after injection, the majority of TA3neo cells were ApopTAG-negative (c) whereas most (>80%) of the remaining TA3sCD44v6-v10 No. 17 cells tested positive for ApopTAG staining (d, arrows). Bar in a–d = 125 μm.
Mentions: To address the fate of TA3sCD44 cells in vivo, and thereby gain insight into the stage of the metastatic process at which cell surface CD44 might be required, we labeled tumor cells with green CMFDA, which allows cell tracing for at least 48 h after injection. Animals injected with the labeled transfectants were killed at 1, 24, and 48 h after injection and lung tissue sections were examined by fluorescence microscopy. Both TA3neo and TA3sCD44 transfectants were found to arrest within lung blood vessels and extravasate into the interstitial stroma within 1 h after injection (Fig. 3 A). Between 1 and 24 h after injection, both TA3neo No. 1 and TA3sCD44v6-10 No. 17 cells were observed to invade lung stroma (Fig. 3 A, a–d, and B, a and b, and data not shown). However, at 48 h, when TA3neo transfectants were forming clusters, only isolated TA3sCD44v6-10 cells were visible (Fig. 3 A, e and f). To determine whether TA3sCD44v6-10 transfectants were undergoing apoptosis within the lung interstitium, TUNEL assays were performed on the tissue sections and TUNEL-positive cells were scored among 100 CMFDA-labeled cells. At 1 and 24 h after injection, minimal TUNEL staining was observed among TA3neo and TA3sCD44v6-10 cells alike (Fig. 3 B, a and b, and data not shown). However, at 48 h, 93 and 88% of the CMFDA-labeled TA3sCD44 cells tested positive for the TUNEL assay in contrast to 10 and 19% of the TA3neo transfectants in two independent experiments (Fig. 3 B, c and d, and data not shown).

Bottom Line: To understand how the hyaluronan receptor CD44 regulates tumor metastasis, the murine mammary carcinoma TA3/St, which constitutively expresses cell surface CD44, was transfected with cDNAs encoding soluble isoforms of CD44 and the transfectants (TA3sCD44) were compared with parental cells (transfected with expression vector only) for growth in vivo and in vitro.However, although parental cells were dividing and forming clusters within lung tissue 48 h following injection, >80% of TA3sCD44 cells underwent apoptosis.Although sCD44 transfectants displayed a marked reduction in their ability to internalize and degrade hyaluronan, they elicited abundant local hyaluronan production within invaded lung tissue, comparable to that induced by parental cells.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathology Unit and MGH Cancer Center, Massachusetts General Hospital, Charlestown Navy Yard, Boston, Massachusetts 02129, USA.

ABSTRACT
To understand how the hyaluronan receptor CD44 regulates tumor metastasis, the murine mammary carcinoma TA3/St, which constitutively expresses cell surface CD44, was transfected with cDNAs encoding soluble isoforms of CD44 and the transfectants (TA3sCD44) were compared with parental cells (transfected with expression vector only) for growth in vivo and in vitro. Local release of soluble CD44 by the transfectants inhibited the ability of endogenous cell surface CD44 to bind and internalize hyaluronan and to mediate TA3 cell invasion of hyaluronan-producing cell monolayers. Mice intravenously injected with parental TA3/St cells developed massive pulmonary metastases within 21-28 d, whereas animals injected with TA3sCD44 cells developed few or no tumors. Tracing of labeled parental and transfectant tumor cells revealed that both cell types initially adhered to pulmonary endothelium and penetrated the interstitial stroma. However, although parental cells were dividing and forming clusters within lung tissue 48 h following injection, >80% of TA3sCD44 cells underwent apoptosis. Although sCD44 transfectants displayed a marked reduction in their ability to internalize and degrade hyaluronan, they elicited abundant local hyaluronan production within invaded lung tissue, comparable to that induced by parental cells. These observations provide direct evidence that cell surface CD44 function promotes tumor cell survival in invaded tissue and that its suppression can induce apoptosis of the invading tumor cells, possibly as a result of impairing their ability to penetrate the host tissue hyaluronan barrier.

Show MeSH
Related in: MedlinePlus